Bachelorproef Chris final

Faculty of Health

Campus Vesalius

Applications of DeNAno particles IL-‐18 Knockoff and Lyophilization

Chris Willems

Academic year 2015-‐2016

Biomedical Laboratory Technology

Pharmaceutical and Biological Laboratory Technology

Promotor Dr. Bradley Messmer, PhD

Laura Ruff, PhD

Co-‐promotor Dr. Stefan Vermeulen

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Acknowledgments First of all I would like to thank Bradley Messmer, PhD, and Laura Ruff, PhD for giving us the

opportunity to follow our internship in their lab. They were always willing to answer all our

questions and help us. This internship was truly a life changing experience.

Furthermore, I would like to thank Dr. Stefan Vermeulen for the guidance with writing this thesis.

I would also like to thank Roberto. This experience would not have been the same, had we not

gone through it together.

Last, I would like to thank my family and friends for all the support they gave me during the

stressful times.

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Table of Contents

Acknowledgments ............................................................................................................. 2

Abstract ................................................................................................................................. 6

Abbreviations ..................................................................................................................... 7

1. Introduction .................................................................................................................... 9

1.1 DeNAno Particles ................................................................................................................. 9 1.1.1 Introduction ..................................................................................................................................... 9 1.1.2 DeNAno particles versus Aptamers ....................................................................................... 9 1.1.3 Biopanning Method ..................................................................................................................... 10

1.2 IL-‐18 DeNano particles .................................................................................................... 13 1.2.1 IL-‐18 Cytokine ............................................................................................................................... 13 1.2.2 IL-‐18 Synthesis ............................................................................................................................. 13 1.2.3 IL-‐18 Receptor and Signaling ................................................................................................. 13 1.2.4 Biological Properties In Cancer ............................................................................................. 14 1.2.5 Levels of IL-‐18 ............................................................................................................................... 15

1.3 Lyophilization ..................................................................................................................... 15 1.3.1 Principle ........................................................................................................................................... 15 1.3.2 Stages of Lyophilization ............................................................................................................ 16

1.4 Research Question ............................................................................................................. 18 1.4.1 DeNano Knockoff from IL-‐18 beads .................................................................................... 18 1.4.2 Lyophilization ................................................................................................................................ 18 1.4.3 Applications of lyophilization ................................................................................................. 18

2. Materials and Methods ............................................................................................. 19

2.1 General .................................................................................................................................. 19 2.1.1 Equipment ....................................................................................................................................... 19 2.1.2 Oligonucleotides ........................................................................................................................... 21 2.1.3 Products ........................................................................................................................................... 21

2.2 DeNAno Particles ............................................................................................................... 22 2.2.1 Ligation ............................................................................................................................................. 22 2.2.2 Rolling circle amplification ...................................................................................................... 24 2.2.3 Staining ............................................................................................................................................. 25 2.2.4 QPCR amplification ..................................................................................................................... 28 2.2.5 Gel electrophoresis ..................................................................................................................... 29 2.2.6 Asymmetric PCR ........................................................................................................................... 30

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2.3 DeNAno particle knockoff by the IL-‐18 cytokine .................................................... 32 2.3.1 Isolation of high-‐copy plasmid DNA from E.coli. ............................................................ 32 2.3.2 QPCR of the isolated DNA ......................................................................................................... 33 2.3.3 Asymmetric PCR of the DeNAno particles ........................................................................ 34 2.3.4 Gel of the asymmetric products ............................................................................................. 34 2.3.5 PCR Purification ........................................................................................................................... 34 2.3.6 Ligation of the S105, S106 and S115 clones ..................................................................... 34 2.3.7 Rolling circle amplification of the S105,S106 and S115 clones ............................... 35 2.3.8 Staining of the S105,S106 and S115 clones ...................................................................... 36 2.3.9 Staining of the S106 clones ...................................................................................................... 36 2.3.10 QPCR of the S106 clones ........................................................................................................ 37 2.3.11 Staining of the S106 clones, repeatability test ............................................................. 37 2.3.12 QPCR of the S106 clones, repeatability test ................................................................... 38 2.3.13 Ligation of DeNAno particle S106-‐61 ............................................................................... 38 2.3.14 Rolling circle amplification of DeNAno particle S106-‐61 ........................................ 39 2.3.15 DeNAno S106-‐61 knockoff by the IL-‐18 cytokine ....................................................... 39 2.3.16 QPCR of the S106-‐61 Total, Supernatant and Pellet samples ................................ 41 2.3.17 S106-‐61 titration with IL-‐18 5min/1hr ......................................................................... 41 2.3.18 QPCR of the S106-‐61 titration Total, Supernatant and Pellet samples .............. 43

2.4 Lyophilization of DeNAno ............................................................................................... 44 2.4.1 Ligation ............................................................................................................................................. 44 2.4.2 Rolling circle amplification ...................................................................................................... 44 2.4.3 Dialysis ............................................................................................................................................. 45 2.4.4 Lyophilization ................................................................................................................................ 46 2.4.5 Staining of the lyophilized samples ..................................................................................... 49 2.4.6 QPCR .................................................................................................................................................. 50

2.5 Applications of lyophilization ........................................................................................ 52 2.5.1 Calculations .................................................................................................................................... 52 2.5.2 Ligation ............................................................................................................................................. 53 2.5.3 Rolling Circle Amplification ..................................................................................................... 54 2.5.4 Dialysis ............................................................................................................................................. 57 2.5.5 Lyophilization ................................................................................................................................ 57

2.6 Different sized DeNAno particles ................................................................................. 59 2.6.1 Ligation S12-‐SA-‐D8 ..................................................................................................................... 59 2.6.2 Rolling Circle Amplification S12-‐SA-‐D8 ............................................................................. 60 2.6.3 Dialysis S12-‐SA-‐D8 ...................................................................................................................... 61

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2.6.4 Lyophilization of S12-‐SA-‐D8 ................................................................................................... 62 2.6.5 Gel of the different sized S12-‐SA-‐D8 particles ................................................................ 62 2.6.6 QPCR with equal concentrations of the different sized products ........................... 62 2.6.7 Ligation of S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10 ............................................................ 63 2.6.8 RCA of the different sized particles ...................................................................................... 63 2.6.9 Dialysis of the different sized particles .............................................................................. 65 2.6.10 Lyophilization of the different sized particles .............................................................. 65 2.6.11 Staining of the different sized particles ........................................................................... 66

3. Results ........................................................................................................................... 69

3.1 DeNAno particle knockoff by the IL-‐18 cytokine .................................................... 69 3.1.1 QPCR Amplification of the isolated DNA ............................................................................ 69 3.1.2 Gel of the QPCR products .......................................................................................................... 69 3.1.3 Gel of the asymmetric products ............................................................................................. 70 3.1.4 Staining of the S105, S106 and S115 clones ..................................................................... 70 3.1.5 Staining of the S106 clones ...................................................................................................... 72 3.1.6 Staining of the S106 clones; Repeatability Test .............................................................. 74 3.1.7 S106-‐61 knockoff by IL-‐18 cytokine ................................................................................... 76 3.1.8 S106-‐61 titration with different concentrations of IL-‐18 .......................................... 80

3.2 Lyophilization ..................................................................................................................... 87 3.2.1 Lyophilization of DeNAno ........................................................................................................ 87 3.2.2 Applications of lyophilization ................................................................................................. 88

4. Discussion ..................................................................................................................... 93

4.1 DeNAno particle knockoff by the IL-‐18 cytokine .................................................... 93 4.2 Lyophilization ..................................................................................................................... 94 4.2.1 Lyophilization of DeNAno particles ..................................................................................... 94 4.2.2 Applications for DeNAno particles ....................................................................................... 94

4.3 Conclusion ............................................................................................................................ 95

References ........................................................................................................................ 96

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Abstract The formation of DeNAno particles starts with the ligation of a 100 bp ssDNA template using a

linker oligonucleotide. The resulting circular template is then used in a rolling circle

amplification (RCA) to form the DeNAno particles. These particles are concatemers that consist of

several hundred copies of the sequence that is complementary to the template. DeNAno partices

are characterized by binding to their target through many low, monovalent affinity interactions,

resulting in overall high avidity.

In the first part of this research study, the ability to quantify the competitive displacement of

DeNAno particles from the beads by the IL-‐18 cytokine was tested. The experiment started with

36 different DeNAno clones. Clones S106-‐61, S106-‐62 and S106-‐64 were the only clones that

stayed bound to the beads long enough during the staining. These clones were tested again in

triplicate to validate the results where clone S106-‐61 showed the best results. In the next step, a

knockoff of the S106-‐61 clone from the IL-‐18 beads with a high concentration of the IL-‐18

cytokine was performed. The amount of release of the DeNAno particles from the beads was

measured at different timepoints. At the “5 min” and “1 hr” timepoints, the release of the DeNAno

particles was high while the background was kept low to increase the sensitivity of the test. A

titration was performed with a dilution series of the IL-‐18 cytokine. The release of the DeNAno

particles was determined after 5 minutes and 1 hour at each concentration of the cytokine. The

“5 min” titration plateaus at a 62,5 nM concentration, while the “1 hour” titration already starts

reaching a plateau at the lower 7,81 nM concentration. The release of DeNAno at the lower

concentrations of the cytokine is negligible after 5 min. This lowers the sensitivity of the test. The

release of DeNAno particles could be differentiated from the background at a 1 nM concentration

of the IL-‐18 cytokine. IL-‐18 has a plasma concentration of 5,55 pM. The sensitivity of the titration

was therefore almost a 200 fold to low to detect the IL-‐18 in human blood samples.

In the second part of this research study, it was attempted to lyophilize DeNAno particles.

Lyophilisation gives us the opportunity to convert DeNaNo particles in solution into a stable

powder form. By dissolving the resulting powder in a smaller volume of water than the original

volume, it is possible to obtain a more concentrated DeNAno solution. On the other hand,

lyophilized samples are easier to store and transport. A staining was performed in order to verify

that the DeNAno particles were intact and were still able to bind to the streptavidin beads.

DeNAno particles S12-‐SA-‐D7 and S12-‐SA-‐D8 were still able to bind after lyophilisation. The

applications of the DeNAno particles were then tested in another three experiments.

Lyophilization in epitubes instead of vials was possible while sample loss was limited. The

lyophilized samples were still able to bind to the streptavidin beads after one month of

incubation at -‐20°C. Different sized DeNAno particles were formed by adjusting the dNTP

concentration during the RCA. These particles couldn’t be visualized at the lower concentrations

during a staining. The DeNAno particles were lyophilized and redissolved in a lower volume of

water. During the staining, only the 3 nmol and 375 pmol dNTP S12-‐SA-‐D7 and S12-‐SA-‐D8 were

able to bind to the streptavidin beads.

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Abbreviations Abbreviation Definition

AcPL Accessory Protein-‐like

APC Antigen Presenting Cell

ASY PCR Asymmetric PCR

Bp Basepairs

BSA Bovine Serum Albumin

Cet Cetuximab

dATP Desoxyadenosine triphosphate

dCTP Desoxycytidine triphosphate

dGTP Desoxyguanosine triphosphate

ΔHs Enthalpy change

DLS Dynamic Light Scattering

DNA Deoxyribonucleic acid

dNTP’s Deoxynucleoside triphosphate

DsDNA Double-‐stranded DNA

DTT Dithiothreitol

dTTP Thymidine triphosphate

HCL Hydrochloride

ICE IL-‐1β-‐ converting enzyme

IFNγ Interferon Gamma

IKK IκB kinase

IL-‐1/IL-‐6 / IL-‐12 / IL-‐18 Interleukin 1/6/12/18

IRAK IL-‐1 Receptor Associated Kinase

kDA KiloDalton

LB Luria-‐Bertani (LB) broth

LPS Lipopolysaccharides

MHC Major Histocompatibility Complex

MW Molecular Weight

NaPhos Sodium Phosphate

NfκB Nuclear Factor Kappa-‐light-‐chain-‐enhancer Of

Activated B Cells

NIK NFκB-‐Inducing Kinase

NK Cells Natural Killer Cells

pAb IgG Polyclonal Antibody Immunoglobulin G

PCR Polymerase Chain Reaction

PG Protein G

Primer F Forward Primer

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Primer R Reverse Primer

qPCR Real-‐Time PCR

RCA Rolling Circle Amplification

Rem Remicade

Rit Rituximab

RNA Ribonucleic acid

Rpm Rounds Per Minute

RT Room Temperature

SA Streptavidin

SN Supernatant

SsDNA Single-‐stranded DNA

TBS Tris Buffered Saline

TBST Tris Buffered Saline Tween-‐20

TNF Tumor Necrosis Factor

TRAF TNF receptor associated factor

TRIS Tris(hydroxymethyl)aminomethane

UW Unwashed

W Washed

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1. Introduction

1.1 DeNAno Particles

1.1.1 Introduction

Affinity reagents, such as monoclonal antibodies, bind their target with high affinity. The problem

with these reagents is that they often can’t distinguish cancer cells from neighboring normal

cells. Furthermore, in order to produce these monoclonal antibodies, a pre-‐identification of the

molecular target is needed1. However, most molecular and cellular in vivo systems use low

affinity, high overall avidity interactions that are still easily reversible. Abreos® Biosciences has

developped a biomolecular affinity reagent that uses hyper-‐avidity instead of single or bi-‐valent

affinity. These DNA nanoparticles (Fig. 1), composed of concatemeric repeats (see 1.1.2), bind to

their target with a low affinity but high overall avidity. This results in a specific and stable but

reversible recognition of polyvalent targets, such as cells or proteins2.

Fig.1. Atomic Force Microscopy image of DeNAno particles3.

1.1.2 DeNAno particles versus Aptamers

In previous studies, aptamers have been used for selection. Aptamers are single-‐stranded DNA or

RNA (ssDNA or ssRNA) molecules that can bind to pre-‐selected targets including proteins and

peptides with high affinity. A similarity between DeNAno particle and aptamer selection is that

both methods do not require a prior knowledge of the target, even when the targets are as

complex as cells. DeNAno particles that are able to bind their target specifically are selected using

a biopanning method that repetitively screens for binding, and uses qPCR to recover the selected

particles4. This method is similar to the selection process of aptamers, which is called SELEX

(systemic evolution of ligands by exponential enrichment). The SELEX method requires a library

of 1012 – 1015 DNA or RNA oligonucleotides, which is incubated with a specific target.

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The oligonucleotides that bind the specific target are then purified and amplified until target

binding oligonucleotides dominate the pool. After the selection process, aptamers are cloned and

sequenced. Aptamers are in general small monovalent fragments of DNA or RNA, typically

smaller than 100 bp, and have high affinity to their target. The difference with DeNAno particles

lies in the fact that DeNAno particles are concatemers, long continuous DNA molecules that

consist of up to several hundreds of copies of the 100 bp DNA fragment linked in series. These

DeNAno fragments do not rely on high affinity, but on low affinity and high overall avidity5.

1.1.3 Biopanning Method

Target-‐specific DeNAno particles are made in four to five selection rounds, using the biopanning

method. This method of selection begins with the ligation of a 100 bp library, which has a 60 base

random region. The ligation product is then amplified by rolling circle amplification (RCA) to

form the DeNAno particles. These particles are then incubated with their specific target coated

beads. The beads are then washed. The attached DeNAno particles are then amplified by qPCR,

followed by asymmetric PCR to form a long single stranded DNA template strand (ssDNA). These

single stranded DNA templates are then re-‐ligated to repeat the cycle (Fig. 2).

1.1.3.1 Ligation

DeNAno particle formation is based on a ssDNA template, composed of 100bp. The template

contains a 60 bp variable region, flanked by a 20 bp specific reigion on both ends. These specific

regions are bound by a 40 bp single stranded complementary linker. A template library consists

of different templates which all contain the same two 20 bp specific regions. Binding of the linker

onto both specific regions makes the ssDNA template circular. The circle formation is acquired

during the annealing step by heating the sample to 95°C. Heating causes the desintegration of

linker that was randomly bound to the template. The specific binding of the complementary

template and linker is acquired by slowly cooling the sample down at a rate of 0,1°C/s from 95°C

to 25°C. T4 DNA Ligase is added after the annealing step to close the circular DNA molecule by

catalyzing the formation of a phosphodiester bond between neighboring 5’ phosphate and 3’

hydroxyl termini6.

1.1.3.2 Rolling Circle Amplification

The linker that was used for the ligation, serves during rolling circle amplification (RCA) as an

initiating primer for Phi29 DNA polymerase to perform the amplification. Phi29 DNA polymerase

is a replicative polymerase obtained from Bacillus subtilis phage phi29 (Φ29), which has high

synthesis properties. This enzyme forms a strand that is complementary to the circular ligation

template. The Phi29 DNA polymerase has an optimal working temperature at 30° C. In order to

stop the amplification, the Phi29 DNA polymerase has to be heat inactivated at 65° C for 10 min.

RCA produces a concatemeric single strand of DNA that is composed of several hundreds of

copies, with a sequence that is complementary to the circular ssDNA template obtained through

ligation. In a regular RCA, the incubation step at 30°C is performed for 30 min. When

amplification of smaller sized DeNAno particles is needed, lower synthesis temperatures or

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shorter incubation times can be used2. Before making the actual DeNAno particles by RCA, a test

has to be performed to determine whether or not circular templates are created during the

ligation step. Therefore, OliGreen is used. OliGreen is a fluorescent dye that binds both ssDNA and

dsDNA. OliGreen binds the amplifying strand in a random fashion but has a preference for

thymine (dTTP), and gives a much higher fluorescent signal when dTTPs are in the sequence..

The higher the amount of copies made, the higher the fluorescent signal is going to be measured

by the microplate reader (TECAN®). Due to the preference OliGreen has for thymine (dTTP), RCA

products with more adenine (dATP) guanine (dGTP) and cytosine (dCTP) will show curves with a

lower slope, even though similar amounts of DeNAno particles are being made7.

Fig. 2. Selection method for DeNAno that bind specific target coated beads. Ligation of a 100 bp library, which has a 60

base random region. The ligation product is then amplified by rolling circle amplification (RCA) to form the DeNAno

particles. These particles are then incubated with their specific target coated beads and washed. The remaining DeNAno

particles are then amplified by qPCR, followed by asymmetric PCR to form an excess of single stranded DNA template strand

(ssDNA). These single stranded DNA templates are re-‐ligated to repeat the cycle2.

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1.1.3.3 Selection and amplification

A library with over 1010 different nanoparticles was used for the first selection against a chosen

target. During the selection, the DeNAno particles are incubated with the beads that are coated

with the target (Fig. 2; 3a) The unbound DeNAno particles are removed in the washing steps

after incubation (Fig. 2; 3b). The bound particles are recovered from the beads, and amplified by

PCR. The forward primer binds to the single stranded DeNAno particles and is elongated by the

DNA polymerase. This complementary template strand is then bound by the reverse primer. Both

strands are amplified during the PCR (Fig. 2; 4a). Only the ssDNA templates are needed in the

rolling circle amplification of the next selection round. An asymmetric PCR with the forward

primer is performed with the 100 bp PCR products. This creates a large amount of ssDNA

templates (Fig. 2; 4b). The asymmetric PCR product is purified to remove excess reagents. The

ssDNA templates are ligated to get circular templates. These templates are used in the rolling

circle amplification of the DeNAno particles of the next selection round.

1.1.3.4 Target specific DeNAno particles

DeNAno that specifically bind to primary human dendritic cells4 and the mouse pancreatic cancer

cell line Panc-‐02 have been selected previously1. During this research project, only DeNAno from

the MJ library that bind specifically to streptavidin were used. Streptavidin and Biotin have a

well-‐characterized system, and it is known for being used in research projects. Streptavidin-‐

specific DeNAno were selected previously.

Fig. 3. Streptavidin-‐specific binding DeNAno. After five rounds of selection four different Streptavidin-‐specific binding DeNAno were found.

The dominant clone was termed SA-‐D8, and the subdominant clone was termed SA-‐D7. G10 was used as a negative control and G10bio was

used as a positive control. This graph shows the binding capacity of all samples to Streptavidin beads. A BSA bead was also used, to show the

specificity of the clones for Streptavidin. The y-‐axis shows the fluorescence in emission intensity, the x-‐axis shows the samples5.

After five selection rounds, these DeNAno were then cloned and sequenced. Four different

streptavidin-‐specific binding DeNAno were found. A dominant clone was found, which was called

SA-‐D8. The subdominant clone that was found was called SA-‐D7. A random clone from the MJ

library was chosen as a negative control, G10neg. By annealing a biotinylated complementary

oligonucleotide to the same random clone, this clone can be used as a positive control, G10bio

(Fig. 3).

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1.2 IL-‐18 DeNano particles

1.2.1 IL-‐18 Cytokine

Interleukin-‐18, formerly known as IFNγ inducing factor, is a member of the IL-‐1 cytokine

superfamily. The cytokines in this superfamily play an important role in the regulation of

immune and inflammatory responses.

1.2.2 IL-‐18 Synthesis

The IL-‐18 gene is found on chromosome 11q22. This gene encodes for a 24 kDa inactive

precursor protein. The Pro-‐IL-‐18 precursor protein consists of a 36 aminoacid propeptide and a

157 polypetide chain8. Pro-‐IL-‐18 is cleaved between the pro-‐peptide and the polypeptide chain

by the endoprotease IL-‐1β-‐ converting enzyme (ICE), also known as caspase-‐1. Caspases are a

family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation9.

The cleavage of Pro-‐IL-‐18 isn’t exclusively executed by ICE as proteinase 3 can also form the

active IL-‐18 protein10. The result is a mature activated 18 kDa Il-‐18 molecule. IL-‐18 expression

has been found in both haemopoietic and non-‐haemopoietic cells such as macrophages, Kupffer

cells, dendritic and Langerhans cells, keratinocytes, osteoblasts, adrenal cortex cells, intestinal

epithelial cells, microglial cells and synovial fibroblasts11. Stimulators such as LPS, exotoxins from

gram-‐positive bacteria and other microbial cytokines induce the production of IL-‐1812.

1.2.3 IL-‐18 Receptor and Signaling

The IL-‐18 receptor is a heterodimeric complex consisting of an α and a β chain. The IL-‐18Rα

chain (originally named IL-‐1 receptor related protein or IL-‐1Rrp) is responsible for the

extracellular binding of the IL-‐18 protein while the IL-‐18Rβ chain (Accessory protein-‐like or

AcPL) is nonbinding and signal transducing10. The IL-‐1Rrp and AcPL genes are found on

chromosome 29.

The IL-‐18Rα binds the IL-‐18 with low affinity, followed by the binding of the IL-‐18Rβ to form a

high affinity heterodimeric complex with the ligand. Each chain contains a signal peptide, an

extracellular segment, a transmembrane region and a cytoplasmic domain. The binding of IL-‐18

to the receptor recruits the IL-‐1 Receptor associated kinases (IRAK). This recruitment requires

an intracellular adapter molecule, MyD88. Phosphorylated IRAK subsequently activates TNF

receptor associated factor 6 (TRAF-‐6), which in turn activates NFκB-‐inducing kinase (NIK). NfκB

consists of IκB and the components p50 and p65. NIK activates IκB kinase (IKK), which in turn

leads to the phosphorylation of IκB. IκB is dissociated from NfκB and is degraded by the

proteasome after ubiquitination. The free NfκB, consisting of the components p50 and p65,

migrates through the nuclear pores to the nucleus where it starts gene transcription (Fig. 4)12.

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Fig. 4. IL-‐18 signal trasnduction. Binding of IL-‐18 to the receptor starts a signaling cascade which ultimately leads to the

migration of the components p50 and p65 to the nucleus where gene transcription takes place12.

1.2.4 Biological Properties In Cancer

1.2.4.1 IFNγ Induction

IFNγ is a 34 kDa homodimeric glycoprotein, produced by Th1 cells, cytotoxic Tc cells and NK

cells. This dimerized cytokine is involved in immunoregulation and tumour control. IFNγ

promotes the recognition of cancer cells by augmenting the expression of MHC molecules.

Furthermore, it stimulates the cytotoxicity of NK cells, T-‐ lymphocytes and macrophages. IL-‐18

stimulates T cell IFNγ production in synergy with IL-‐129.

1.2.4.2 Expression of Fas Ligand

Fas ligand is a transmembrane protein from the TNF family. Binding of FAS-‐L to the FAS-‐receptor

induces apoptosis in the Fas-‐R presenting cell. IL-‐18 up-‐regulates FAS-‐L expression on NK and

Th1 cells and therefore contributes to the induction of apoptosis in FAS-‐R presenting cells12.

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1.2.4.3 Differentiation T Helper Cells

T helper cells (Th cells) can be organised into two groups with different cytokines and immune

functions (Table 1).

Th1 cells Th2 cells

Cytokine

production

Il-‐2, IL-‐18, IFNγ, TNFα IL-‐4, IL-‐5, IL-‐6, IL-‐10, IL-‐13

Immune function Inflammatory and cell-‐mediated

immune responses

Humoral and allergic

responses

Table 1. Th1 and Th2 comparison9.

The differentiation of Th0 cells to Th1 or Th2 cells is determined by the nature of the antigen, the

type of APC and the local cytokine production. The synergy between IL-‐18 and IL-‐12 leads to the

differentiation of naive Th0 cells to Th1 cells but not Th2 cells. IL-‐18 also activates T cells to

produce IL-‐2 and TNFα, important cytokines for the destruction of cancer cells9.

1.2.5 Levels of IL-‐18

To design a quantitative detection method of IL-‐18, it is important to know what de levels of IL-‐

18 in serum are. The detection method is only useful if the sensitivity is high enough to

differentiate the IL-‐18 value from the background. In a study measuring the circulating IL-‐18 in

patients with Leukaemia, healthy persons showed levels of plasma IL-‐18 between 50 and 150

pg/ml. Patients with Leukemia showed elevated levels of 200-‐1200 pg/ml12.

1.3 Lyophilization

1.3.1 Principle

The freeze-‐drying or lyophilization process is a method to remove water from frozen materials13.

It is based on the principle of sublimation. Sublimation is the transition of a substance directly

from the solid phase to the gas phase without passing through an intermediate liquid phase. In

the ideal case, this results in the retention of the microstructure of the frozen material, which

promotes rapid and complete rehydration of the solid sample14. Sublimation is an endothermic

phase transition that occurs at temperatures and pressures below a substance’s triple point in its

phase diagram (Fig. 5)15. The triple point of H2O is 273.16 K or 0.1°C 16. The material to be dried

is first frozen and then subjected under a high vacuum to heat, so that frozen liquid vaporizes,

leaving only solid, dried components of the original liquid13.

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Fig. 5. Phase diagram of H2O. Sublimation of water occurs when using pressure below 6.11 mbar and temperature below 0°

C 13.

1.3.2 Stages of Lyophilization

1.3.2.1 Snap Freezing

Snap freezing is the process in which samples, in this case RCA products, are lowered to

temperatures below 70°C very rapidly using liquid nitrogen. This step is necessary to reduce the

chance of ice crystals being formed by water present in the sample during the freezing process17.

Most samples that are used for freeze-‐drying are eutectics. Eutetic solutions freeze at lower

temperatures than the surrounding water. When the aqueous suspension is cooled, changes

occur in the solute concentrations of the product matrix. During the cooling process the water is

separated from the solutes as it changes to ice. This creates more concentrated areas of solute.

These pockets of concentrated materials have a lower freezing temperature than the water. Only

when the eutectic temperature is achieved, will the suspension be frozen. The eutectic

temperature is specific for each solution, and is the temperature at which not only the water, but

every component present in the solution is frozen. Small pockets of unfrozen material remaining

in the product expand and can compromise the structural stability of the freeze-‐dried product,

hence should be avoided as much as possible13.

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1.3.3.2 Primary drying

The process of primary drying starts by reducing the pressure in the lyophilizer to a level, which

is lower than the vapor pressure of ice at the sublimation front in the product to promote

sublimation of ice and transfer of water vapor to the condenser13. Sublimation starts by adding

energy in a quantity equal to the heat of sublimation of ice ΔHS14. Chamber pressure exceeding

the vapor pressure reduces the sublimation rate by reducing the pressure gradient between the

sublimation front and the chamber. This results in prevention of mass transfer. On the other

hand when chamber pressure is too low, the rate of heat transfer to the product is limited.

Fig. 6. Schematic figure of heat and mass transfer in a freeze-‐dryer. Addition of heat to the frozen sample causes the

conversion of the solid ice into vapor. This vapor is transferred to the condenser coil through a vacuum produced by the

pump13.

Due to the fact that the mass transfer starts at the top of the frozen product, a three-‐layer system

is formed. This three-‐layer system consists of the dry cake, the sublimation front and the

remaining frozen solution (Fig. 6). As the dried layer increases, it becomes a greater barrier for

mass transfer out of the vials13. The sublimation rate is equal to the pressure difference divided

by the resistance. The total resistance to mass transfer consists of three components: firstly the

thickness of the dry cake, which increases as the drying proceeds, secondly the partially inserted

stopper in the vial and thirdly the resistance to flow vapor from the drying chamber to the

condenser in the freeze-‐dryer itself 14.

1.3.3.3 Secondary drying

After primary drying there is still around 20% of the solid weight left of unfrozen water. This is

removed by diffusion during the secondary drying. Diffusion is the molecular flow from a region

of high concentration to a region of lower concentration14. In the area where the ice has already

been removed, desorption of water from the dry cake occurs. This process of secondary drying

already starts in the primary drying phase13.

18

1.4 Research Question

1.4.1 DeNano Knockoff from IL-‐18 beads

In this project we want to test the ability to quantify the competitive displacement of DeNAno

particles from the beads by the IL-‐18 protein. We therefore used DeNAno particles, bound on

beads which are coated with IL-‐18 antibodies. We then performed competitive displacement in

the presence of IL-‐18 in the solution. Our aim was to determine the lowest concentration of the

IL-‐18 cytokine, able to dissociate DeNAno particles from the beads, hence, the binding sensitivity

of the test.

1.4.2 Lyophilization

Lyophilisation gives us the opportunity to convert DeNaNo particles in solution into a stable

powder form. By dissolving the resulting powder in a smaller volume of Hypure Molecular

Biology Grade water than the original volume, it is possible to obtain a more concentrated

DeNAno solution. This can be applied in vivo when there is a limit on the volume that can be

administered to the organism.

1.4.3 Applications of lyophilization

We will be doing three different experiments simultaneously to optimalize lyophilization of

DeNAno particles:

• Comparison of the lyophilization of the samples in vials versus the

lyophilization in 1,5 ml tubes. The vials can only be sealed with Parafilm,

while the 1,5 ml tubes have a screwcap, which would be easier to seal the

samples with.

• Testing whether it is possible to store the lyophilized samples long term in

the freezer without reinstating them in Hypure Molecular Biology Grade

water.

• We have tried to make particles with different sizes by modifying the RCA

protocol. The smallest particles could be visualised with the DLS machine

but couldn’t be differentiated from the background during the staining. We

may be able to visualize the particles by concentrating them after

lyophilization and testing the concentration with Nanodrop.

19

2. Materials and Methods

2.1 General

2.1.1 Equipment

Equipment Specification Company

Balance Classic plus Mettler Toledo, Greifensee,

Switzerland

Centrifuge Allegra X-‐15R Centrifuge Beckman Coulter, IN, USA

Centrifuge Microfuge 18 Centrifuge Beckman Coulter, IN, USA

Centrifuge Microfuge 22R Centrifuge Beckman Coulter, IN, USA

Conicals Olympus

50 ml, 15 ml

Genesee Scientific, CA, USA

Dialysis cassettes Slide-‐A-‐Lyzer G2 Dialysis

cassettes 10K, 100–500 μL,

0,1-‐0,5 ml, 0,5-‐3,0 ml.

Thermo Scientific, MA, USA

Filter unit Steriflip EMD Millipore, MA, USA

Fridge 4°C VWR, PA, USA

Fridge/freezer 4°C/-‐20°C VWR, PA, USA

Glass cylinder 500 ml VWR, PA, USA

Glass Erlenmeyer Pyrex

1000 ml, 500 ml

Corning Incorporated, NY, USA

Gloves TITAN E-‐GEN

Powder-‐free latex

examination gloves


Greiner plate Costar

Storage plate, no lid

96-‐ well V bottom

Corning Incorporated, NY, USA

Heat block Digital heat block VWR, PA, USA

Heat and stir plate Super-‐nuova multi-‐place Thermo Scientific, MA, USA

Ice machine / Hoshizaki America, Inc., GA,

USA

Kimwipe (delicate task

wipers)

/ Kimtech, Kimberly-‐Clark

Professional, GA, USA

Laboratory film / Parafilm, WI, USA

Microcentrifuge tubes Seal-‐Rite

Natural tubes: 1.5 ml, 2 ml

USA Scientific, FL, USA

Micropipet 0.1 -‐ 2.5 µl, 0.5-‐10 µl, 2 -‐ 20 µl,

20 -‐ 200 µl, 100 -‐ 1000 µl

Eppendorf, Hamburg,

Germany

20

Micropipet tips Olympus

Barrier tips: 10 µl, 20 µl, 200

µl, 1000 µl


Micropipet tips Low Binding, Racked and

Stacked, Sterile, 200 µl


Minicentrifuge Fisher Scientific, NJ, USA

Minicentrifuge Galaxy Mini VWR, PA, USA

Minicentrifuge Mini Mouse Delville Scienfitic, NJ, USA

Multichannel pipet 0.5 -‐ 10 µl, 8 tips Eppendorf, Hamburg,

Germany

Multichannel pipet 10 -‐ 100 µl: 12 tips, 30 -‐ 300

µl: 12 tips

Biohit, Helsinki, Finland

Optical Cap, 8x Strip / Agilent Technologies, CA, USA

Optical PCR tubes, 8x

Strip

0.2 ml 8-‐well Agilent Technologies, CA, USA

PCR strip tubes 0.2 ml 8-‐well Genesee Scientific, CA, USA

Pipets Olympus

Serological pipets: 10 ml, 5 ml


Plastic cylinder Nalgene

250 ml, 100 ml

Thermo Scientific, MA, USA

Sealing films Seal-‐Plate

Films, non-‐sterile


Thermal cycler Applied Biosystems

2720 thermal cycler

Life Technologies, CA, USA

Thermal cycler DNA Engine thermal cycler Bio-‐Rad, CA, USA

Vacuum-‐driven filter

system

Olympus

PES membrane, 0.22 µm


Vortex Analog Vortex Mixer VWR, PA, USA

Vortex Analog Vortex Mixer Thermo Scientific, MA, USA

96-‐well white VWR plate Disposable skirted white plate

for QPCR

VWR, PA, USA

21

2.1.2 Oligonucleotides

Oligonucleotide Specification Company

MJ Alexa Fluor 647 probe

Sequence: 5’/CCA AAA AGC

AAG GAT CCA ACT C/3’

MW: 7,710.6 g/mol

Integrated DNA Technologies,

IA, USA

MJ biotinylated probe

Sequence: 5’/GGA TCC AAC

TCA ACG TCA CC/3’

MW: 6,424.4 g/mol


IA, USA

2.1.3 Products

Product Product number Lot number Company

Bovine Serum

Albumin (BSA)

S15898 148303 Thermo Scientific, MA,

USA

DNA Erase L8917/ L9042-‐L (refill)

065K0507 Sigma Aldrich, MO,

USA

Hypure Molecular

Biology Grade water

SH3053803 AZK193756 GE Lifesciences, UK

MgCL2 M0304 M030417D1301 Teknova, CA, USA

Milli-‐ Q water

/ / EMD Millipore, MA,

USA

20X TBS, pH 7,4 T1680 T168015D1501 Teknova, CA, USA

1M Tris-‐Hcl pH 8,5 T5085 T508507K1201 Teknova, CA, USA

Tween-‐20 103168 7682K MP Biomedicals, CA,

USA

22

2.2 DeNAno Particles

2.2.1 Ligation

2.2.1.1 Materials

2.2.1.1.1 Oligonucleotides


MJ linker Sequence: GGT GAC GTT GAG

TTG GAT CCT TGC TTT TTG

GAA CTC CTG CT

MW: 12,635.2 g/mol


IA, USA

S12-‐SA-‐D7 template Sequence: 5’/GGA TCC AAC

TCA ACG TCA CCT GCG TCG

GTG CCG GTT GCG TAT AGA

CTT TAA TGC GAG TGA AGT

GTA CTA GAG AAA AAC AAA

GCA GGA GTT CCA AAA AGC

AA/3’

MW: 31,383.3 g/mol


IA, USA

S12-‐SA-‐D8 template Sequence: 5’/GGA TCC AAC

TCA ACG TCA CCA ATT ATC

ACT ATC TCA CTG CGT CGT

TTC GAC AGT GTC ATA TAA

AAC TAG TCA TAT ATT CTA


AA/3’

MW: 31,048.1 g/mol


IA, USA

S13-‐G10 template Sequence: 5’/GGA TCC AAC

TCA ACG TCA CCT CAG TTA

AGT TTG ATG GTA TAG TTG

AAA GTT GTG TCT AGT TCG

GCC CAT ATA TAT TCT TAA


AA/3’

MW: 31,261.2 g/mol


IA, USA

23

2.2.1.1.2 Products

Product Product Number Lot Number Company

Hypure Molecular

Biology Grade water


Pierce DTT

(Dithiothreitol)

20291 QA211997A Thermo Scientific, MA,

USA

T4 DNA Ligase M0202L 1081409 New England BioLabs

Inc., MA, USA

10X T4 DNA Ligase

reaction buffer

B0202S 0021412 New England BioLabs

Inc., MA, USA

2.2.1.2 Methods

Reagents Volume 1X (μl)

PCR water 41,5 or volume to bring total to 50

T4 Buffer, 10X 5

DTT, 500 mM 0,5

Library primer/ Asymmetric PCR

product, 0,5 μM

1 (or to equal 0,5-‐1 pmols)

(Use 1 pmol for asymmetric products)

Linker, 1 μM 1,5 ( or to equal 1,5 pmols)

T4 Enzyme 0,5

• Mix ingredients 1 to 5 together in a PCR tube. Remember that each library uses a

different linker. Make a mastermix of ingredients 1 to 3 if you’re doing multiple ligations.

• Spin down

• Put the tubes in a thermal cycler and run the “Anneal” program comprising of the

following steps: 95°C for 2 min, ramp cool to 25 °C at a rate of 0,1 °C/sec. An anneal

program may also consist of heating to 95°C for 2 min and then turn off the thermal

cycler to cool to room temperature.

• Add 0,5 μl T4 enzyme to each tube.

• Incubate at room temperature for 5 min or overnight at 4°C.

24

2.2.2 Rolling circle amplification

2.2.2.1 Materials


Deoxynucleotide

(dNTP) solution

set

N0446S 0521403 New England BioLabs

Inc., MA, USA

Hypure Molecular

Biology Grade

water


Phi29 DNA

Polymerase

M0269L 0131312 New England BioLabs

Inc., MA, USA

10X Phi 29 DNA

Polymerase

Buffer


Inc., MA, USA

Pierce DTT

(Dithiothreitol)

20291 QA211997A Thermo Scientific,

MA, USA

Quant-‐iT OliGreen

ssDNA Reagent

O7582 / Life Technologies, CA,

USA

2.2.2.2 Methods

Reagent Volume 1X (μl)

PCR Water 38,7

Phi29 Buffer, 10X 5

dNTPs, 10 mM 0,3

DTT, 500mM 0,5

Oligreen, 200X 0,25

Sample (Ligation) 5

Phi29 Enzyme 0,25

• Set temperature at 30°C (Instrument/Heating/30,Set,On) on the microplate analyser.

• Prepare a mastermix of ingredients 1 to 5. Make enough for a negative reaction as well.

• Aliquot 5 μl of sample into a 96-‐well white VWR plate.

• Check that the microplate analyser is ready to go.

• Add Phi29 enzyme to the mastermix. This enzyme works quickly, so move along.

• Add 45 μl of mastermix to each well of the white VWR plate.

• Mix the samples in the wells with a multichannel pipet set to 30 μl.

• Put the plate in the microplate analyser and read.

• Graph your samples on the excel sheet as it is running.

25

• If your curve shows a slope with a minimal value of 1 and the R2 approaches 1, go and

make your particles with the following changes: a) Replace Oligreen with PCR water

b) Set heat blocks to 30°C and 65°C and use 1,5 ml tubes (or use PCR

tubes and the PCR cycler,” RCA30” program)

c) Your RCA should run at 30°C for 30 min (this is adjustable, depending

on how big you want your particle to be) and 65°C for 10 min (Heat

inactivates the enzyme). Heat at 65°C for 15 min if you are using larger

volumes in the heat blocks.

• If you need to label the RCA with a fluorescent or biotinylated probe ( For staining): Add

1,5 μl probe/50 μl RCA reaction ( 1/10 molar ratio of 1 μM probe). Mix and run the

“Anneal” program. Wait 1 hour before using or put in the fridge overnight.

2.2.3 Staining

2.2.3.1 Materials


Bovine Serum

Albumin (BSA)

S15898 148303 Thermo Scientific,

MA, USA

Cetuximab 1176105A5 IMF355 Bristol-‐Myers Squibb,

NY, USA

MgCL2 M0304 M030417D1301 Teknova, CA, USA

Milli-‐ Q Water


USA

Polyclonal Human

IgG

31154 PE1853834 Thermo Scientific,

MA, USA

Protein G beads 88847 PD202855 Thermo Scientific,

MA, USA

Remicade 10022309 EHL63015P1 Janssen Biotech, NJ,

USA

Rituximab 10134058 590472 Genentech, CA, USA

Sodium Azide 5% 7144.8-‐16 1211163 Ricca Chemical, TX,

USA

Sodium

Phosphate Buffer,

0,5M pH 7,5

BB-‐2324 D11X1 Boston Bioproducts,

MA, USA

Streptavidin

Beads

S1420S 0181309 New England BioLabs

Inc., MA, USA

20X TBS, pH 7,4 T1680 T168015D1501 Teknova, CA, USA

Tween-‐20 103168 7682K MP Biomedicals, CA,

USA

26

2.2.3.1.1 Preparation of 500 ml TBS 1% BSA solution

• 1% BSA m/v = 1g/100ml

à Weigh 5g BSA

• Transfer 475 ml Milli-‐Q water in a 500ml flask. Add 25 ml of the 20X TBS and 5g BSA.

Mix. Filtrate when dissolved with the vacuum-‐driven filter system and store at 4°C.

2.2.3.1.2 Preparation of 500 ml TBST 1% BSA 0,05 % Tween-‐20 10 mM MgCl2 solution

• Tween-‐20: 5% . V1 = 0,05% . 500ml

à V1= 5ml of the 5% Tween-‐20 stock

• 1% BSA m/v = 1g/100ml

à 5g BSA

• 10 mM MgCl2 in 500 ml solution = 5 mmol MgCl2

Stock 1M MgCL2

1 mol/L = 1 mmol/mL

à 5mmol = 5 ml of the 1M MgCL2 stock

• TBS: 20X . V1 = 1X . 500ml

à V1= 25 ml of the 20X TBS stock

• Transfer 465 ml Milli-‐Q water in a 500ml flask. Add 25 ml of the 20X TBS, 5 ml of the 5%

Tween-‐20 stock, 5ml of the 1M MgCl2 stock and 5g BSA. Mix. Filtrate when dissolved

with the vacuum-‐driven filter system and store at 4°C.

2.2.3.1.3 Preparation of the magnetic beads

2.2.3.1.3.1 Preparation of 20 mM Na Phos pH 7,5 buffer

• Stock concentration: 0,5M

20 mM . 5000 μl = 500 mM . V1

à V1 =200 μl stock 0,5 M Na Phos pH 7,5 + 4800 μl Milli-‐Q water.

2.2.3.1.3.2 Preparation of 50 mM Na Phos pH 7,5 buffer

• Stock concentration: 0,5M

50 mM . 50 μl = 500 mM . V1

à V1 =5 μl stock 0,5 M Na Phos pH 7,5 + 45 μl Milli-‐Q water.

2.2.3.1.3.3 Calculations

• Remicade 10,0 ng/ μl Make 50 μl Remicade Beads

Rituximab 10,0 ng/ μl Make 300 μl Rituximab Beads

IL-‐6 1,064 ng/ μl Make 20 μl IL-‐6 Beads



Cetuximab 2,0 ng/ μl Make 50 μl Cetuximab Beads

27

• For 100 μl of beads, use 60 μg of Ab.

Remicade: 50 μl à 30 μg

We need 3 μl and add 47 μl 20mM Na Phos pH 7,5 buffer.

Rituximab: 300 μl à 180 μg


Cetuximab: 50 μl à 30 μg


IL-‐6: 20 μl à 12 μg

We need 11,5 μl and add 8,5 μl 50mM Na Phos pH 7,5 buffer.

IL-‐12: 20 μl à 12 μg


IL-‐18: 30 μl à 18μg


2.2.3.1.3.4 Coating of the beads with the target antibodies

• Wash the magnetic Protein G beads 3 times with 20 mM Na Phos pH 7,5 buffer.

• Protein G beads can bind 60 μg IgG/mg of beads. (Beads come in a solution of 10mg/ml,

so 600 μg IgG can be bound to 1 ml of beads). So for 100 μl of beads, use 60 μg of

antibody. Make the antibody dilutiion in 20 mM Na Phos pH 7,5 buffer (same volume as

beads ).

• Resuspend the beads in the antibody dilution. Incubate at RT for 2hr or 4°C overnight.

Flick to mix occasionally.

• Wash the beads 3 times with 20 mM Na Phos pH 7,5 buffer.

• Resuspend in PBS 0,02 % Na Azide.

2.2.3.2 Methods

• Pre-‐block the wells that you are going to use with 200 μl TBS 1% BSA for 30 min.

• Add magnetic beads to 1 well of the Greiner plate or 1,5 ml tube. 1-‐2 μl beads/sample ( +

extra).

• Wash with TBST 10 mM MgCL2 3 times. Put the plate on a 96-‐well magnet to collect the

beads each time.

• Resuspend the beads in TBST 10 mM MgCL2,, 1-‐2 μl beads/ 50 μl volume. Aliquot 50 μl

TBST 10 mM MgCL2 + beads int each well.

• Add 50 μl RCA into each well.

• Incubate 1 hour at RT.

• Add 100 μl TBST 10 mM MgCL2, put on magnet to collect the beads.

• Wash with 200 μl TBST 10 mM MgCL2 4 times, magnet.

• Resuspend in 50 μl TBS 1% BSA.

• Transfer the samples to a 96-‐well white VWR plate.

28

2.2.4 QPCR amplification

2.2.4.1 Materials



MJ Primer F Sequence: /5’ Phos/GGA TCC

AAC TCA ACG TCA CC

MW: 6111, 0 g/mol


IA, USA

MJ Primer R Sequence: 5’/ TGC TTT TTG

GAA CTC CTG CT/3’

MW: 6065,0 g/mol


IA, USA

2.2.4.1.2 Products


Hypure Molecular

Biology Grade

water


Hemo Klentaq

Buffer


Inc., MA, USA

SybrGreen S7563 1135054 Thermo Scientific,

MA, USA

Hemo Klentaq

Enzyme


Inc., MA, USA

Deoxynucleotide

(dNTP) solution

set


Inc., MA, USA

2.2.4.2 Methods

• Turn on the PCR Stratagene cycler. Turn on the lamp to warm up (20 min) and label your

template.

• Thaw reagents on the chill box.

• Make dilutions of the staining samples in TBS 1% BSA.

• Spray/ wipe PCR hood with DNA Erase. Put open a 2ml tube and PCR tubes in the PCR

hood and UV for 5 min.

29

Reagent Volume 1X (μl)

PCR water 32,5

Hemo Klentaq Buffer, 5X 10

dNTPs, 10 mM 1

SybrGreen, 100X 0,5

Primer F, 10 μM 1,5

Primer R, 10 μM 1,5

Hemo Klentaq Enzyme 1

Sample (Staining) 2

• Bring all reagents except enzyme and sample to the PCR hood.

• Make a mastermix of the first 6 ingredients. Keep tubes in chill box.

• Bring enzyme in a chill box to the PCR hood. Add enzyme to the mastermix. Put enzyme

away.

• Aliqout the mastermix in the PCR tubes.

• Take the PCR tubes to the bench. Mix the samples. Add samples to the PCR tubes. Mix.

• Spin Down.

• Put samples in the PCR cycler. Try to avoid the outer ring of wells if you can.

• Thermal Profile Setup: 1 cycle at 95°C for 10 min

25 cycles at 95°C/20s; 55°C/30s; 68°C/1min

1 cycle at 95°C/1min; 55°C/30s; 95°C/30s

• Plate setup: Well type: Unknown

Collect fluorescence data: Sybr

2.2.5 Gel electrophoresis

2.2.5.1 Materials


Agarose 20-‐102 LF45130044 Genesee Scientific, CA,

USA

25/100 Bp DNA

Ladder

D-‐1020 1203H Bioneer Corporation,

Korea

Gelcode Red Dye 41003 14G1002 Biotium, CA, USA

Log 2 ladder N3200L / New England BioLabs

Inc., MA, USA

0,75X TBE Buffer / / /

30

2.2.5.2 Methods

• Set up plate, comb, and stand. Make sure it is level.

• Prepare 2% agarose by adding 4g agarose in 200 ml 0,75X TBE buffer.

• (Preparation of a 1% agarose gel)

à 0,6g agarose in 60 ml TBE buffer

• Melt in the microwave

• Cool on bench

• Add gelcode Red Dye (1 μl Red Dye / 20 ml gel) = 10 μl Red Dye added

• Pour gel into tray and let solidify

• Add gel to running tray, and fill with 0,75X TBE buffer.

• Mix 5 μl of QPCR with 1 μl of loading buffer.

• Load the samples into the wells. Also load a ladder. (1,6 μl ladder in the small wells/ 2 μl

ladder in the wider wells)

• Run at 100V for 45-‐60 min.

• Put on UV Transilluminator and take picture.

2.2.6 Asymmetric PCR

2.2.6.1 Materials



MJ Primer F Sequence: Sequence: /5’

Phos/GGA TCC AAC TCA ACG

TCA CC

MW: 6111, 0 g/mol


IA, USA

2.2.6.1.2 Products


Deoxynucleotide

(dNTP) solution

set


Inc., MA, USA

Hemo Klentaq

Buffer, 5X


Inc., MA, USA

Hemo Klentaq

Enzyme


Inc., MA, USA

Hypure Molecular

Biology Grade

Water


31

2.2.6.2 Methods

2.2.6.2.1 Asymmetric PCR

Reagent Volume 1X (μl) (For 50 μl) Volume 1X (μl) (For 100 μl)

PCR water 31,5 63

Hemo Klentaq Buffer, 5X 10 20

dNTPs, 10mM 1 2

Primer F, 10 μM 4,5 9

Hemo Klentaq Enzyme 1 2

Sample/QPCR 2 4

• Make at least 100 μl of asymmetric product for each sample.

• Do in the PCR hood, same as setup as QPCR.

• Make a mastermix of the first 5 ingredients.

• Add 4 μl sample to the PCR tubes.

• Run the “Asypcr” program.

• After the PCR is complete, clean up the PCR product with the QIAgen PCR Purification kit.

Elute with 30 μl buffer EB.

• Nanodrop the samples.

2.2.6.2.2 PCR purification

• Add 6 volumes Buffer PB to 1 volume of the PCR reaction and mix. Mix in an Epitube

before adding to column.

• If the colour of the mixture is orange or violet, add 5 μl 3 M sodium acetate, pH 5.0 to 50

μl of PCR sample and mix. The color of the mixture will turn yellow.

• Place a QIAquick column into a vacuum manifold.

• To bind DNA, load the samples to the QIAquick columns and apply vacuum to the

manifold until all the samples have passed through the column. After the samples have

passed through the column, switch off the vacuum source.

• To wash, add 0.75 ml Buffer PE to the QIAquick column and incubate for 5 min. Apply

vacuum.

• Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.

• To elute the DNA, add 30 μl buffer EB (10 mM Tris·Cl, pH 8.5), to the center of the

QIAquick membrane, let the column stand for 1 min, and then centrifuge.

• If the purified DNA is to be analysed on a gel, add 1 volume of Loading Dye to 5 volumes

of purified DNA. Mix the solution by pipetting up and down before loading the gel.

32

2.3 DeNAno particle knockoff by the IL-‐18 cytokine

2.3.1 Isolation of high-‐copy plasmid DNA from E.coli.

• Cultivate and harvest bacterial cells

1. Use 1-‐5 ml of a saturated E.coli LB culture, pellet cells in a standard

benchtop microcentrifuge for 30s at 11000 x g. Discard the supernatant

and remove as much liquid as possible (depp on a paper).

• Cell lysis

1. Add 250 μl Buffer A1. Resuspend the cell pellet completely by vortexing

or pippetting up and down. Make sure no cell clumps remain before

addition of Buffer A2.

2. Add 250 μl Buffer A2. Mix gently by inverting the tube 6-‐8 times. Do not

vortex to avoid shearing of genomic DNA. Incubate at RT for up to 5 min

or until the lysate appears clear.

3. Add 300 μl Buffer A3. Mix thoroughly by inverting the tube 6-‐8 times.

Do not vortex to avoid shearing of genomix DNA.

• Clarification of lysate

1. Centrifuge for 5 min at 11000 x g at RT. Repeat this step in case the

supernatant is not clear.

• Bind DNA (use the vacuum manifold)

1. Place a NucleoSpin Plasmid/ Plasmid (Nolid) Column in a 2ml collection

tube. Decant the supernatant from the previous step onto the column.

Place on the vacuum manifold.

• Wash silicia membrane

1. Add 600 μl Buffer A4. Put the vacuum on.

• Dry silica membrane

1. Put the column in a collection tube en centrifuge for 2 min at 110000 x

g. Discard the collection tube.

• Elute DNA

1. Place the column in a 1,5 ml epitube and add 50 μl Buffer AE. Incubate

for 1 min at RT. Centrifuge for 1 min at 11000 x g.

• Measure the concentrations with Nanodrop.

33

2.3.2 QPCR of the isolated DNA

The isolated DNA is amplificated with QPCR.

2.3.2.1 Materials

• See section 2.2.4.1

• The project started with 36 different clones, divided between Roberto and I.

Target Library Selection Number

IL-‐18 Abr03 S105-‐50

IL-‐18 Abr03 S105-‐52

IL-‐18 Abr03 S105-‐53

IL-‐18 Abr03 S105-‐58

IL-‐18 MJ S105-‐61

IL-‐18 MJ S105-‐62

IL-‐18 MJ S105-‐63

IL-‐18 MJ S105-‐64

IL-‐18 MJ S105-‐65

IL-‐18 MJ S105-‐66

IL-‐18 MJ S105-‐68

IL-‐18 MJ S105-‐71

Cetuximab MJ S105-‐74





2.3.2.2 Methods


Reagent Volume 1X (μl) Volume 46X (μl)

PCR water 32,5 1495


dNTPs, 10 mM 1 46

SybrGreen, 100X 0,5 23


Primer R, 10 μM 1,5 69


Sample (Staining) 2

34

2.3.3 Asymmetric PCR of the DeNAno particles

SsDNA template strands are generated by asymmetric PCR using only the forward primer.

2.3.3.1 Materials


2.3.3.2 Methods



PCR water 31,5 2520


dNTPs, 10mM 1 80

Primer F, 10 μM 4,5 360


Sample/QPCR 2

2.3.4 Gel of the asymmetric products

Check if all the asymmetric samples are 100 bp.

2.3.4.1 Materials


2.3.4.2 Methods


2.3.5 PCR Purification

• All excess reagents are removed.

• See section 2.2.6.2.2.

• Nanodrop the samples.

2.3.6 Ligation of the S105, S106 and S115 clones

2.3.6.1 Materials


Nanodrop

concentration (ng/

μl)

Molecular

weight ( g/mol)

Concentration

(μM)

Volume for

1,5 pmol (μl)

MJ linker 14,2 12635,2 1,12 1,34

Abr03 linker 12,0 12524 0,958 1,57

35

2.3.6.2 Methods


• The 18 purified asymmetric PCR products are made circular by ligation. These cicular

asymmetric products will function as a template for the formation of the DeNAno

particles. There are 4 clones from the Abro3 library and 14 clones from the MJ library.

Make a mastermix of PCR Water, T4 buffer and DTT. Split the mastermix in 662,10 μl for

the MJ library and 220,7 μl for the Abr03 library.

Reagents Volume 1X (μl) Volume 20X (μl)

PCR water 38,86 777,2

T4 Buffer, 10X 5 100

DTT, 500 mM 0,5 10

Library primer/ Asymmetric

PCR product, 0,5 μM

± 3,68

MJ Linker, 1 μM 1,34

Abr03 Linker, 1 μM 1,57

T4 Enzyme 0,5 10

2.3.7 Rolling circle amplification of the S105,S106 and S115 clones

• Single stranded DeNAno concatomers are made of the 18 clones. These concatomers are

complementary to the template oligonucleotide.

2.3.7.1 Materials


2.3.7.2 Methods


• Test RCA


PCR water 38,7 812,7

Phi29 Buffer, 10X 5 105

dNTPs, 10 mM 0,3 6,3

DTT, 500mM 0,5 10,5

Oligreen, 200X 0,25 5,25

Sample (Ligation) 5

Phi29 Enzyme 0,25 5,25

36

• Real RCA


PCR water 38,95 817,95


dNTPs, 10 mM 0,3 6,3

DTT, 500mM 0,5 10,5

Sample (Ligation) 5


2.3.8 Staining of the S105,S106 and S115 clones

2.3.8.1 Materials


2.3.8.2 Methods


2.3.9 Staining of the S106 clones

2.3.9.1 Materials


2.3.9.2 Methods


• Add magnetic beads to 1 well of the Greiner plate or 1,5 ml tube. 1-‐2 μl beads/sample ( +

extra).


beads each time.

• Resuspend the beads in TBST 10 mM MgCL2,, 1-‐2 μl beads/ 50 μl volume. Aliquot 50 μl

TBST 10 mM MgCL2 + beads int each well.






• Make a 5 μl aliquot from each well. This is the Total sample.

• Put a Sealplate film on the plate and incubate 1 hr at RT.

• Pull the beads down and collect the supernatant.

• Resuspend the beads in 200 μl TBST 1% BSA 10 mM MgCl2 buffer, magnet.

• Wash 3x 200 μl TBST 1% BSA 10 mM MgCl2 , magnet.

37


• Make a 5 μl aliquot from each well. This is the Resuspended beads (RB) sample.

• Make a 5 μl aliquot from the supernatants. This is the Supernatant (SN) sample.

• Make 1:20 dilutions of each aliquot by adding 95 μl TBS 1% BSA.

• Make 1:5 dilutions by adding 20 μl of each diluted sample to 80 μl TBS 1% BSA.

2.3.10 QPCR of the S106 clones

2.3.10.1 Materials


2.3.10.2 Methods


• We will select the clones that show the following results:

1. The Ct of the "Total “ sample comes up first.

2. The Ct of the “Resuspended beads” sample is close to the Ct of the

“Total” sample.

3. The Ct of the “Supernatant” sample is a couple Ct’s lower than the Ct of

the “Total” sample.

These results suggest that DeNAno stays on the beads during the 1 hour incubation and

the following washing steps.


PCR water 29,5 1475


dNTPs, 10 mM 1 50





Sample 5

2.3.11 Staining of the S106 clones, repeatability test

2.3.11.1 Materials


2.3.11.2 Methods


38

2.3.12 QPCR of the S106 clones, repeatability test

2.3.12.1 Materials


2.3.12.2 Methods



PCR Water 29,5 1622,5


dNTPs, 10 mM 1 55

SybrGreen, 100X 0,5 27,5

Primer F, 10 μM 1,5 82,5

Primer R, 10 μM 1,5 82,5


Sample (Staining) 5

2.3.13 Ligation of DeNAno particle S106-‐61

2.3.13.1 Materials


Nanodrop

concentration

(ng/ μl)

Molecular

weight ( g/mol)

Concentration

(μM)

Volume

for 1,0

pmol (μl)

Volume for

1,5 pmol

(μl)

MJ linker 14,2 12635,2 1,12 / 1,34

S106-‐61 7,4 31000 2,39 4,18 /

2.3.13.2 Methods


• Make 100 μl ligation.


PCR water 38,48 76,96

T4 Buffer, 10X 5 10

DTT, 500 mM 0,5 1

Library primer/

Asymmetric PCR product,

0,5 μM

4,18 8,36

Linker, 1 μM 1,34 2,68

T4 Enzyme 0,5 1

39

2.3.14 Rolling circle amplification of DeNAno particle S106-‐61

2.3.14.1 Materials


2.3.14.2 Methods


• Test RCA




dNTPs, 10 mM 0,3 1,2

DTT, 500mM 0,5 2

Oligreen, 200X 0,25 1

Sample (Ligation) 5

Phi29 Enzyme 0,25 1

• Real RCA

• Use 95 μl Ligation.


PCR water 38,95 779


dNTPs, 10 mM 0,3 6

DTT, 500mM 0,5 10

Sample (Ligation) 5

Phi29 Enzyme 0,25 5

2.3.15 DeNAno S106-‐61 knockoff by the IL-‐18 cytokine

2.3.15.1 Materials



Human Recombinant

IL-‐18

B003-‐2 075 MBL, MA, USA

2.3.15.2 Methods

• Make TBST 1% BSA 10 mM MgCl2 buffer ( 500ml – washing and 50 ml – dilutions only).

• Block a plate with 200 μl TBST 1% BSA 10 mM MgCl2 , >30 min at RT ( Block 5 wells for

each sample ( a total of 10 wells for each clone) and 1 to wash the beads).

40

• Incubate 12,5 μl S106-‐61 RCA + 0,5 μl IL-‐18 Ab beads + 37 μl TBST 1% BSA 10 mM MgCl2

for 1 hr at RT. ( Need 2 wells for every clone) (1st well).

• Wash 1x 175 μl TBST 1% BSA 10 mM MgCl2 , magnet ( Incubations on magnet are done

for the same amount of time throughout), transfer to new wells.

• Wash 3x 200 μl TBST 1% BSA 10 mM MgCl2 , magnet, transfer to new wells ( 2nd-‐3rd

blocked wells for these washing steps; Don’t need to change wells every single time).

• Resuspend in 50 μl TBST 1% BSA 10 mM MgCl2 or 50 μl 1μM IL-‐18 protein in TBST 1%

BSA 10 mM MgCl2, move this to your 4th blocked well ( 1 well each for each clone +/-‐IL-‐

18)

• Take 5 μl aliquot and dilute in 45 μl TBST 1% BSA 10 mM MgCl2. Save “Total” sample.

Take 5 μl aliquot of beads only and dilute in 45 μl TBST 1% BSA 10 mM MgCl2.

• Incubate. Take 5 μl supernatant aliquots at 5 min, 30 min, 1hr, 2 hr. (Dilute in 45 μl TBST

1% BSA 10 mM MgCl2) Resuspend the sample then put on magnet before taking aliquot

each time. Keep plate covered to minimize evaporation.

• At last time point, remove all supernatant.

• Resuspend beads in 200 μl TBST 1% BSA 10 mM MgCl2.

• Magnet, remove and discard supernatant, resuspend in 200 μl TBST 1% BSA 10 mM

MgCl2. Move to new well (5th well).

• Repeat previous wash step.

• Resuspend the beads in 200 μl TBST 1% BSA 10 mM MgCl2.

• Take 5 μl aliquot and dilute in 45 μl TBST 1% BSA 10 mM MgCl2. This is the “Pellet”

sample.

• Dilute “Total” samples an additional 1:10 in TBST 1% BSA 10 mM MgCl2 (1:100 total).

• Dilute “Supernatant” samples an additional 1:10 in TBST 1% BSA 10 mM MgCl2 (1:100

total).

• Dilute “Pellet” samples an additional 1:12,5 in TBST 1% BSA 10 mM MgCl2 (1:100 total).

• Repeat this protocol 3 times to validate the results.

Adjustments 3th experiment

• Execute the protocol but implement following changes:


• Resuspend beads in 200 μl TBST 1% BSA 10 mM MgCl2 and take a 5 μl aliquot. Dilute in

45 μl TBST 1% BSA 10 mM MgCl2. This is the “Resuspended 1” sample.

• Magnet, remove and discard supernatant, resuspend in 200 μl TBST 1% BSA 10 mM

MgCl2 . Move to new well ( 5th well). Take a 5 μl aliquot. Dilute in 45 μl TBST 1% BSA 10

mM MgCl2. This is the “Resuspended 2” sample.

• Repeat previous wash step and take a 5 μl aliquot. Dilute in 45 μl TBST 1% BSA 10 mM

MgCl2. This is the “Resuspended 3” sample.

• Resuspend the beads in 200 μl TBST 1% BSA 10 mM MgCl2.

41

• Take 5 μl aliquot and dilute in 45 μl TBST 1% BSA 10 mM MgCl2. This is the “Pellet”

sample.

• Dilute “Total” samples an additional 1:10 in TBST 1% BSA 10 mM MgCl2 (1:100 total).

• Dilute “Supernatant” samples an additional 1:10 in TBST 1% BSA 10 mM MgCl2 (1:100

total).

• Dilute “Resuspended” and “Pellet” samples an additional 1:12,5 in TBST 1% BSA 10 mM

MgCl2 (1:100 total).

2.3.16 QPCR of the S106-‐61 Total, Supernatant and Pellet samples

2.3.16.1 Materials


2.3.16.2 Methods


Reagent Volume 1X (μl) Volume 27X (μl) Volume 33X (μl) (including

Resuspended Samples)

PCR water 29,5 796,5 973,5

Hemo Klentaq

Buffer, 5X

10 270 330

dNTPs, 10 mM 1 27 33

SybrGreen, 100X 0,5 13,5 16,5

Primer F, 10 μM 1,5 40,5 49,5

Primer R, 10 μM 1,5 40,5 49,5

Hemo Klentaq

Enzyme

1 27 33

Sample (Staining) 5

2.3.17 S106-‐61 titration with IL-‐18 5min/1hr

2.3.17.1 Materials



Human Recombinant

IL-‐18

B003-‐2 075 MBL, MA, USA

2.3.17.1.1 Preparation IL-‐18 protein dilutions

• Make 8 threefold dilutions of the 1 μM stock.

• Il-‐18 protein stock concentration: 5,55 µM.

• 150 μl . 1 μM = V1 . 5,55 μM

42

à V1 = 27 μl stock + 122,97 μl TBST 1%BSA 10mM MgCl2

• 150 μl . 500 nM = V1 . 1 μM

à V1 = 75 μl 1 μM stock + 75 μl TBST 1%BSA 10mM MgCl2

• 150 μl . 250 nM = V1 . 500 nM

à V1 = 75 μl 500 nM stock + 75 μl TBST 1%BSA 10mM MgCl2

• 150 μl . 125 nM = V1 . 250 nM


• 150 μl . 62,5 nM = V1 . 125 nM


• 150 μl . 31,25 nM = V1 . 62,5 nM

à V1 = 50 μl 62,5 nM stock + 100 μl TBST 1%BSA 10mM MgCl2

• 150 μl . 15,63 nM = V1 . 31,25 nM


• 150 μl 7,81 nM = V1 . 15,63 nM


• 150 μl . 3,91 nM = V1 . 7,81 nM


• 150 μl . 1,95 nM = V1 . 3,91 nM


• 150 μl . 0,98 nM = V1 . 1,95 nM


2.3.17.2 Methods

• Make TBST 1%BSA 10mM MgCl2 buffer (500ml-‐-‐washing and 50ml-‐-‐dilutions only)

• Wash 6 µl IL-‐18 beads in an epitube 4 times. Resuspend the beads in 412,5 µl TBST

1%BSA 10mM MgCl2 and aliquot 37,5 µl in each well.

• Block plate with 200ul TBST 1% BSA 10mM MgCl2 (or TBS 1% BSA), ≥30min at RT.

• Incubate 12.5ul IL-‐18 clone + 0.5ul IL-‐18 ab beads + 37ul TBST 1% BSA 10mM MgCl2 for

1hr at RT. (need 10 wells for every clone) (1st well)

• Wash 1x 175ul TBST 1%BSA 10mM MgCl2, magnet (incubations on magnet done for

same amount of time throughout), transfer to new wells

• Wash 3x200ul TBST 1%BSA 10mM MgCl2, magnet, transfer to new wells (2nd-‐3rd

blocked wells for steps 4-‐5. Don’t need to change wells every single time)

• Resuspend in the different concentrated solutions:

1. 50 µl 0,98 nM IL-‐18 protein in TBST 1%BSA 10mM MgCl2





43



8. 50 µl 125 nM IL-‐18 protein in TBST 1%BSA 10mM MgCl2

9. 50 µl 250 nM IL-‐18 protein in TBST 1%BSA 10mM MgCl2

10. 50 µl 1 µM IL-‐18 protein in TBST 1%BSA 10mM MgCl2

11. 50 µl TBST 1%BSA 10mM MgCl2

• Move this to your 4th blocked well.

• Take 5ul aliquot from all 11 wells and dilute in 45ul TBST 1%BSA 10mM MgCl2. Save

‘Total” sample.

• Incubate. Take 5ul supernatant aliquots at 5min, 1hr (dilute in 45ul TBST 1%BSA 10mM

MgCl2, as in #7). Resuspend sample then put on magnet before taking aliquot each time.

Keep plate covered to minimize evaporation. Save “Supernatant” samples for each

dilution.


• Resuspend beads in 200ul TBST 1%BSA 10mM MgCl2

• Magnet, remove supernatant (discard), resuspend in 200ul TBST 1%BSA 10mM MgCl2.

Move to new well (5th well)

• Repeat #11 (don’t need to transfer wells)

• Resuspend beads in 200ul TBST 1%BSA 10mM MgCl2.

• Take 5ul aliquot and dilute in 45ul TBST 1%BSA 10mM MgCl2. Save “Pellet” sample.

• Dilute samples an additional 1:10 in TBST 1%BSA 10mM MgCl2 (1:100 total)

2.3.18 QPCR of the S106-‐61 titration Total, Supernatant and Pellet samples

2.3.18.1 Materials


2.3.18.2 Methods



PCR water 29,5 1770


dNTPs, 10 mM 1 60





Sample (Staining) 5

44

2.4 Lyophilization of DeNAno

We are testing the lyophilization of samples S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10.

2.4.1 Ligation

2.4.1.1 Materials


2.4.1.2 Methods


2.4.2 Rolling circle amplification

2.4.2.1 Materials


2.4.2.2 Methods


• Test RCA




dNTPs, 10 mM 0,3 1,2

DTT, 500mM 0,5 2


Sample (Ligation) 5

Phi29 Enzyme 0,25 1

• We are going to make 450 μl RCA instead of 200 μl for the Real RCA. During dialysis the

volume of each sample will increase. After dialysis we will split the total volume of each

sample in half . One half of the volume will be lyophilized, the other half will be used as a

control.


PCR water 350,55 1402,2


dNTPs, 10 mM 2,7 10,8

DTT, 500 mM 4,5 18

Ligation 45

Phi29 Enyme 2,25 9

45

• We are using 1,5 ml tubes instead of PCR tubes because of the bigger volume that each

sample has.

• Set the heat blocks to 30°C and 65°C

• Run the RCA at 30°C for 30 min and 65°C for 15 min.

2.4.3 Dialysis

2.4.3.1 Materials

Products Product number Lot number Company

Milli-‐ Q water


USA

Slide-‐A-‐Lyzer G2

Dialysis cassettes

10K, 0,1-‐0,5 ml

88250 OH189048 Thermo Scientific, MA,

USA

1M Tris-‐Hcl pH 8,5 T5085 T508507K1201 Teknova, CA, USA

2.4.3.1.1 Preparation of the dialysis buffer

• The dialysis buffer that we make is the same solution in which we want to lyophilize our

samples.

• Each sample has a volume of 450 μl. We need to make a buffer at least 300 times the

volume of the sample: 300 x 450 μl = 135 ml buffer for each sample. That makes a total

of 405 ml buffer for all samples together.

• We are using a 10 mM Tris-‐Hcl pH 8,5 buffer, made from a 1M Tris-‐Hcl pH 8,5 stock.

• 405ml . 10mM = V1 . 1000mM

à V1 = 4,05 ml 1M Tris-‐Hcl pH 8,5 + 400,95 ml Milli-‐Q water.

2.4.3.2 Methods

• Hydrate the membrane

1. Remove the cassette from its protective pouch. To prevent membrane

contamination, handle the casette by the plastic frame only. Do not

touch the membrane with ungloved hands. The cassette may be placed

upright on its bottom end on a flat surface.

2. Immerse the cassette in dialysis buffer for 2 minutes. It may be

necessary to hold the cassette under the surface for the hydration step

as the air inside the cassette may cause it to float sideways.

3. Remove cassette from buffer. To remove excess buffer, gently tap the

cassette on a paper towel. Leave the cassette in the buffer until you are

ready to load the sample. Write the sample name on the top of the cap.

46

• Add Sample

1. Open the cassette by gently twisting the cap counter-‐clockwise until it

stops (± 45° angle) and then gently pulling out the cap.

2. Add the sample using a 200 μl pipettor, slowly withdrawing the pipette

while dispensing. Do not overload the cassette.

3. Remove the excess air in the cassette by simultaneously pressing the

membrane gently on both sides using your gloved thumb and forefinger

and inserting the cap.

4. Insert cap and lock by gently twisting it clockwise.

• Dialyze Sample

1. Float the cassette vertically in the dialysis buffer and stir gently to avoid

creating a vortex that might pull the casette down in contact with the

stir bar. Stir at 50 rpm on the magnetic stirplate in the coldroom.

2. Dialyze overnight at RT or 4°C ( Cold room). Replace the dialysis buffer

and dialyze overnight. Replace the dialysis buffer and dialyze overnight

one last time. Use the dialysis buffer at a total of at least 300 times the

volume of the sample during the course of the dialysis procedure.

• Remove the sample

1. Remove the cassette from the buffer. To remove excess buffer, gently

tap the cassette on a paper towel. Turn the cassette upside down and

tap again.

2. Open the cassette by gently twisting the cap counter-‐clockwise until it

stops (± 45° angle) and then gently pulling out the cap.

3. Use 100 μl gel-‐loading tips to retrieve the sample by slowly aspirating

while inserting the pipette toward the bottom of the cassette.

4. Transfer each sample into a 1,5ml tube. Determine the exact volume of

each sample.


2.4.4.1 Snap freezing

2.4.4.1.1 Materials


Epitubes with

screwcaps

72.692 / Sarstedt, DEU

Lyophilization

vials

C4000-‐2W 13221175 Thermo Scientific,

MA, USA

Lyophilization

vial caps

B7800-‐1 00149132 Thermo Scientific,

MA, USA

47

2.4.4.1.2 Methods

• Spray the caps and vials with ethanol to desinfect them.

• Aliqout the exact volume of each sample into a vial.

• Before we can lyophilize the samples, we have to snap freeze them first in liquid

nitrogen.

• Use the blue protection gloves and the face shield before opening the tank.

• Put the vials in a cardboard box with holes in the bottom in order to let the liquid flow

out after being submerged.

• Check the nitrogen level with a measuring stick.

• Use the hose on the wall to fill up the tank if the level is too low. Cover the tank with the

blanket while filling. Rechcek the level. The level should be at least 10.

• Put the cardboard box on the bottom shelf of one of the racks. Don’t forget to secure the

boxes with the pin.

• Submerge the rack and close the tank.

• Wait until the samples solidify.

• Keep the samples frozen in dry ice until you use them in the lyophilizator.

2.4.4.2 Lyophilization

2.4.4.2.1 Materials


Kimwipe (delicate

task wipers)

34155 FL416928 Kimtech, Kimberly-‐

Clark Professional,

GA, USA

Fast-‐Freeze®

Flasks

18-‐1912-‐07 75409-‐00 Labconco, MO, USA

Lyophilizer 7934027 071178960 C Labconco, MO, USA

2.4.4.2.2 Methods

• Clean the inside of the glass cover and the tank to prevent ice formation.

• Close the cover.

• Start the lyophiliser.

• Press “ Auto Refrigeration”.

• Wait until the display shows: ± 0,500 mbar

-‐70°C

• Put paper towels on top of the glass cover to steady the glass container. Wipe the inside

of the glass container twice with a Kimwipe to clean out all the liquid.

• Remove the caps from the sample tubes.

• Seal the opening of the tubes with a folded Kimwipe and wrap the Kimwipe around the

opening of the tube to cover it.

48

• Cut the elastic of a glove off and use it to hold the Kimwipe firmly in place.

• Fill the glass container with Kimwipes.

• Place the samples upright in the container without the openings touching eachother.

• Put a maximum of 5 vials together. Separate two layers of vials with a couple of

Kimwipes.

• Firmly press the cap on the glass container.

• Firmly press the glass tube on top of the cap into one of the openings of the drying

chamber.

• Rotate the knob 180° so that the slant is facing to the container. Turn the knob slowly in

order to expose the samples slowly to the difference in pressure.

• Let lyophilize overnight.

• Close the valve by rotating the knob slowly 180° counter-‐clockwise. Slowly expose the

samples to the atmospheric pressure.

• Remove the container.

• Open the valve slowly by rotating the knob 180° clockwise to release the vacuum.

• Stop the lyophiliser.

• Clean the container and the containertop three times with ethanol after removing the

samples and Kimwipes.

2.4.4.3 Nanodrop

• Resuspend the lyophilized samples in a tenth of the starting volume. The samples will be

ten times more concentrated. The concentration of the lyophilized samples and the

control samples must be the same in order to do the staining. The concentrations of both

samples is analysed with Nanodrop. The lyophilized samples are then diluted to have

the same concentration as the control samples.

• Compare the nanodrop readings with the concentration which would be expected of the

RCA under perfect conditions in order to get an idea of how good the RCA works and

how much sample might be lost during dialysis. Each sample has a bigger volume after

dialysis. Compensate for the volume difference in order to calculate the original

concentration.

• Calculation perfect RCA concentration

1 pmol template oligo in 50 μl ligation

Use 5 μl ligation in RCA

± 600 copies 100 bp/ RCA in a 30 min reaction

MW 100 bp ≅ 31000 g/mol

à 1 pmol template in 50 μl = 0,1 pmol template in 5 μl

à 0,1 pmol x 600 copies = 60 pmol in 50 μl RCA

à 31000 g/mol x 60 pmol = 1860000 pg = 1860 ng

à 1860 ng/ 50 μl = 37,2 ng/ μl

49

• Results Nanodrop

Dialysis (Control) Lyophilization (1/10th of the original volume)

S12-‐SA-‐D7 14,6 ng/μl 132,3 ng/μl

S12-‐SA-‐D8 15,0 ng/μl 152,0 ng/μl

S13-‐G10 15,6 ng/μl 134,2 ng/μl

• Comparison of the Nanodrop results with the theoretical ideal RCA concentration shows

that all the samples are in the same ballpark.

2.4.5 Staining of the lyophilized samples

2.4.5.1 Materials


2.4.5.2 Methods


• The concentration of the lyophilized samples and the control samples must be the same

in order to do the staining.

• Concentration dialysed control samples

See section 2.4.4.3

• Calculation dilutions lyophilized samples

S12-‐SA-‐D7

132,3 ng/μl . V1 = 14,6 ng/μl

à V1 = 5,52 μl sample + 44,48 μl Hypure Molecular Biology Grade water

S12-‐SA-‐D8

152,0 ng/μl . V1 = 15,0 ng/μl


S13-‐G10

134,2 ng/μl . V1 = 15,6 ng/μl


• Label the RCA’s with the MJ v1 Alexa probe and the biotinylated probe.

50 μl S12-‐SA-‐D7 LYO 1,112 μl MJ v1 Alexa probe

50 μl S12-‐SA-‐D7 DIA 1,112 μl MJ v1 Alexa probe

50 μl S12-‐SA-‐D8 LYO 1,112 μl MJ v1 Alexa probe

50 μl S12-‐SA-‐D8 DIA 1,112 μl MJ v1 Alexa probe

50 μl S13-‐G10 LYO 1,112 μl MJ v1 Alexa probe

50 μl S13-‐G10 DIA 1,112 μl MJ v1 Alexa probe

50 μl S13-‐G10Bio LYO 1,112 μl MJ v1 Alexa probe 2,200 μl Biotinylated probe

50 μl S12-‐G10Bio DIA 1,112 μl MJ v1 Alexa probe 2,200 μl Biotinylated probe

50

2.4.6 QPCR

2.4.6.1 Materials


2.4.6.2 Methods


• The quantitative plate read results were diificult to differ from the background. A QPCR

reading was performed to get a more sensative read.

• Use a magnet to pull the beads down in the optical tubes that were used for the

quantitative plate read.

• Remove the supernatant which contains magnesium. Samples S12-‐SA-‐D7 and S12-‐SA-‐D8

showed a lot of aggregates during the washing steps of the staining. Aggregates give a

aberrant result in a QPCR. DeNAno particles need magnesium to bind to the beads. The

aggregates will disappear by removing the supernatant with magnesium.

• Make a 1/1000 dilution. Resuspend the beads in 200 μl TBS 1% BSA ( instead of 50 μl).

This is the first ¼ dilution.

• Incubate for 30 min . Mix the samples with a pipettor after 15 min to remove most

aggregates.

• Resuspend the samples.

• Take a 4 μl aliquot from each sample and add 96 μl TBS 1% BSA.

Take a 10 μl aliquot from each of the dilution and add 90 μl TBS 1% BSA.

• Prepare a series of standards in order to determine the concentration of each sample.

Make 7 tenfold dilutions of the 1 ng/μl standard. Use a 100bp plasmid stock with known

concentration.

Concentration of the S84-‐5 stock: 102,9 ng/μl

102,9 . V1 = 10 ng/μl . 100 μl

à V1 = 9,718 μl + 90,28 μl TBS 1% BSA

10 ng/ μl . V1 = 1 ng/μl . 100 μl

à V1 = 10 μl + 90 μl TBS 1% BSA

The 1 ng/μl stock is the first standard. Aliquot 90 μl TBS 1% BSA in the remaining 7 PCR

tubes. Add 10 μl 1 ng/μl stock in the first PCR tube. Mix. Add 10 μl of this first dilution to

the next PCR tube. Repeat this for the remaining PCR tubes.

1ng/μlà10-‐1 ng/μl à10-‐2 ng/μl à10-‐3 ng/μl à10-‐4 ng/μl à10-‐5 ng/μl à10-‐6 ng/μl

à 10-‐7 ng/μl

51


PCR water 29,5 708


dNTPs, 10 mM 1 24





Sample (Staining) 5

Aliquot 45 μl mastermix in each well. Add 5 μl sample and mix.

• Protocol and settings QPCR machine

See section 2.2.4.2

52

2.5 Applications of lyophilization

We are doing the three experiments simultaneously.

2.5.1 Calculations

1. Comparison of the lyophilization with the vials versus the 1,5 ml epitubes

Vials Epitubes

S12-‐SA-‐D7

200 μl of RCA

A1

200 μl of RCA

A2

S12-‐SA-‐D8

200 μl of RCA

B1

200 μl of RCA

B2

2. Long term storage of the lyophilized samples

Vials

S12-‐SA-‐D7 200 μl of RCA

A3


B3

3. Lyophilization of different sized particles

Lyophilization

X Y Z


A4

200 μl of RCA

A5

200 μl of RCA

A6


B4

200 μl of RCA

B5

200 μl of RCA

B6

Dialysis control

X Y Z


A7

200 μl of RCA

A8

200 μl of RCA

A9


B7

200 μl of RCA

B8

200 μl of RCA

B9

X= 30min / 3nmol dNTPs (Standard RCA)

Y = 30 min / 93,75 pmol dNTPs

Z = 5 min / 93,75 pmol dNTPs

53

2.5.2 Ligation

2.5.2.1 Materials


2.5.2.2 Methods

2.5.2.2.1 Nanodrop and concentration calculations

Sample Concentration (ng/μl) MW (g/mol)

MJv1 Linker 14,2 12635

S12-‐SA-‐D7 17,1 31383

S12-‐SA-‐D8 9,5 31048

• µμM = !"#$ !! =

(!"!! ! !"!)

!!"#

• MJv1 Linker: (!",!!"!! ! !"

!)

(!"#$% !!"#)

= 1,12 μM

• S12-‐SA-‐D7: !",!!"!! ! !"

!

!"!#! !!"#

= 0,545 µμM

• S12-‐SA-‐D8: (!,! !"!"!! ! !"

!)

(!"#$% !!"#)

= 0,306 μM

Calculate the average template-‐ and linkervolume to determine the watervolume.

Template !,!"! !! ! !,!"# !!

! = 0,426 μM

1pmol template: 0,426 μM = ! !"#$! !!

à Add 2,35 μl template / 50 μl reaction.

Linker

1,5 pmol linker: 1,12 μM = !,! !"#$! !!

à Add 1,34 μl linker / 50 μl reaction

• Calculation of the exact templatevolumes

S12-‐SA-‐D7: 0, 545 μM = !"#$%! !!

à 1,8 μl sample / 50 μl reaction = 7,2 μl sample / 200 μl

reaction

S12-‐SA-‐D8: 0,306 μM = !"#$%! !!

à 3,3 μl sample / 50 μl reaction = 13,2 μl sample / 200 μl

reaction

54

2.5.2.2.2 Ligation

Reagents Volume 1X (μl) ( For 90

μl)

Volume 1X (μl) ( For 400

μl)

PCR water 72,56 322,5

T4 Buffer, 10X 9 40

DTT, 500 mM 0,9 4


product, 0,5 μM

± 4,23 (1pmol)

Linker, 1 μM ± 2,41 (1,5 pmol) 10,72

T4 Enzyme 0,9 4

• Make a mastermix of PCR water, T4 Buffer, DTT and Linker.

• Aliquot 84,87 μl mastermix in four seperate tubes. Add 3,24 μl S12-‐SA-‐D7 template to

tube 1 and 2 and 5,94 μl S12-‐SA-‐D8 template to tube 3 and 4.

• Spin down

• Put the tubes in a thermal cycler and run the “ Anneal” program comprising of the

following steps: 95°C for 2 min, ramp cool to 25 °C at a rate of 0,1 °C/sec. An anneal

program may also consist of heating to 95°C for 2 min and then turn off the thermal

cycler to cool to room temperature.

• Add 1 μl T4 enzyme to each tube.

• Incubate at room temperature for 5 min or overnight at 4°C.

2.5.3 Rolling Circle Amplification

2.5.3.1 Materials

See section 2.2.2.1

2.5.3.2 Methods


2.5.3.2.1 Test RCA




dNTPs, 10 mM 0,3 1,5

DTT, 500mM 0,5 2,5

Oligreen, 200X 0,25 1,25

Sample (Ligation) 5


55

2.5.3.2.2 Real RCA

Reagent Volume 1X (μl) ( For 200 μl) Volume 20X (μl)

PCR water 155,8 3116


dNTPs, 10 mM 1,2

DTT, 500mM 2 40

Sample (Ligation)

Phi29 Enzyme 1

• Make a mastermix with PCR water, Phi29 buffer and DTT

• Aliquot 1066,8 μl mastermix in tube MM1

Aliquot 711,2 μl mastermix in tube MM2

Aliquot 1422,4 μl mastermix in tube MM3

• Modify the protocol in order to get different sized particles:

§ 30min / 3nmol dNTPs (Standard RCA)

§ 30 min / 93,75 pmol dNTPs

§ 5 min / 93,75 pmol dNTPs

• Make dilutions of the different dNTPs concentrations in order to add 1,2 μl dNTPs to

each 200 μl reaction.

Concentration dNTPs stock: 10mM

§ Standard 50 μl reaction : ! !"#$!,! !!

= 10 mM

§ Modified 50 μl reaction: !",!" .!"!! !"#$

!,! !! = 0,3125 mM

10mM . V1 = 0,3125 mM . 100 μl

à V1 = 3,125 μl 10mM dNTPs stock + 96,875 μl Hypure Molecular

Biology Grade water

• Tube setup

Content information see section 2.5.1

Content

Tube 1 A1/A2/A3

Tube 2 B1/B2/B3

Tube 3 A4/A7

Tube 4 B4/B7

Tube 5 A5/A8

Tube 6 B5/B8

Tube 7 A6/A9

Tube 8 B6/B9

56

• Add 7,2 μl standard dNTPs to tube MM1.

Add 4,8 μl modified dNTPs to tube MM2.

Add 9,6 μl modified dNTPs to tube MM3.

• Add 6 μl Phi29 Enzyme to tube MM1.

Add 4 μl Phi29 Enzyme to tube MM2.

Add 8 μl Phi29 Enzyme to tube MM3.

• Add the following volumes of ligation to the appropraite tubes.

Ligation

Tube 1 60 μl S12-‐SA-‐D7








• Aliquot 540 μl MM1 into tubes 1 and 2.

Aliquot 360 μl MM2 into tubes 3 and 4.

Aliquot 360 μl MM3 into tubes 5,6,7 and 8.

§ Because the RCA in tube 7 and 8 will only run for 5 min at 30°C, it is important

to add and mix the mastermix next to the heatblocks in order to save as much

time as possible.

• Set heat blocks to 30°C and 65°C and 95°C.

30°C 65°C 95°C

Tube 1 30 min 15 min

Tube 2 30 min 15 min

Tube 3 30 min 5 min

Tube 4 30 min 5 min

Tube 5 30 min 5 min

Tube 6 30 min 5 min

Tube 7 5 min 5 min

Tube 8 5 min 5 min

57

2.5.4 Dialysis

2.5.4.1 Materials




samples.

• We have two samples with a volume of 600 μl and 6 samples with a volume of 400 μl.

We need to make a buffer at least 300 times the volume of the sample:

§ 300 x 600 μl = 180 ml buffer. That makes a total of 360 ml buffer for tube 1 and

2.

§ 300 x 400 μl = 120 ml buffer. That makes a total of 360 ml buffer for tubes 3,4, 5

and a total of 360 ml buffer for tubes 6,7,and 8.


• 360 ml . 10mM = V1 . 1000mM

à V1 = 3,60 ml 1M Tris-‐Hcl pH 8,5 + 356,4 ml Milli-‐Q water.

2.5.4.2 Methods



2.5.5.1 Materials

• See sections 2.4.4.1.1 and 2.4.4.2.1

2.5.5.2 Methods

• See sections 2.4.4.1.2 and 2.4.4.2.2

• Determine the volume of each tube after dialysis and divide it into vials for

lyophilization.

Volume after Dialysis (μl)

Tube 1 1608

Tube 2 1374

Tube 3 600

Tube 4 760

Tube 5 645

Tube 6 706

Tube 7 700

Tube 8 628

58

• Sample setup see section 2.5.1

Sample Volume Sample Volume

A1 536 μl from Tube 1 B1 458 μl from Tube 2




A5 322,5 μl from Tube 5 B5 353 μl from Tube 6



A8 322,5 μl from Tube 5 B8 353 μl from Tube 6


59

2.6 Different sized DeNAno particles

We are testing sample S12-‐SA-‐D8.

2.6.1 Ligation S12-‐SA-‐D8

2.6.1.1 Materials


2.6.1.2 Methods


2.6.1.2.1 Nanodrop and concentration calculations

Sample Concentration (ng/μl) MW (g/mol)

MJv1 Linker 14,2 12635

S12-‐SA-‐D8 9,5 31048

• 𝜇𝑀 = !"#$ !! =

(!"!! ! !"!)

!!"#

• MJv1 Linker: (!",!!"!! ! !"

!)

(!"#$% !!"#)

= 1,12 μM

• S12-‐SA-‐D8: (!,! !"!"!! ! !"

!)

(!"#$% !!"#)

= 0,306 μM

1pmol template: 0,306 μM = ! !"#$! !!

à Add 3,26 μl template / 50 μl reaction.

Linker

1,5 pmol linker: 1,12 μM = !,! !"#$! !!

à Add 1,34 μl linker / 50 μl reaction

2.6.1.2.2 Ligation


PCR water 39,4 78,8

T4 Buffer, 10X 5 10

DTT, 500 mM 0,5 1


product, 0,5 μM

3,26 6,52

Linker, 1 μM 1,34 2,68

T4 Enzyme 0,5 1

60

2.6.2 Rolling Circle Amplification S12-‐SA-‐D8

We are making three different sized particles of S12-‐SA-‐D8 by changing the dNTP’s concentration

for each 30min reaction.

2.6.2.1 Materials


2.6.2.3 Methods


2.6.2.3.1 Calculations

• dNTPs stock = 10 mM

• 30 min 3 nmol S12-‐SA-‐D8 ! !"#$!,! !!

= 10mM

• 30 min 375 pmol S12-‐SA-‐D8 !"# !!"!! !"#$

!,! !! = 1,25 mM

V1 . 10mM = 50 μl. 1,25 mM

à V1 = 6,25 μl 10mM stock + 43,75 μl Hypure Molecular Biology Grade water

• 30 min 93,8 pmol S12-‐SA-‐D8 !",! !!"!! !"#$

!,! !! = 0,3127 mM

V1 . 10mM = 50 μl. 0,3127 mM


• Dilute the dNTPs ten times and add 3 μl of the 1,0 mM solution to the PCR tubes instead

of 0,3 μl of the 10mM solution. Volumes lower than 1 μl aren’t pippetable accurately.

V1 . 10mM = 3 μl. 1,0 mM

à V1 = 0,3 μl + 2,7 μl Hypure Molecular Biology Grade water

2.6.2.3.2 Test RCA


PCR water 36 144


dNTPs, 1 mM 3

DTT, 500mM 0,5 2


Sample (Ligation) 5

Phi29 Enzyme 0,25 1

61

2.6.2.3.3 Real RCA

• 3 samples + 1 negative RCA

Reagent Volume 1X (μl) ( For 200 μl) Volume 5X (μl)

PCR water 145 725


dNTPs, 1 mM 12

DTT, 500mM 2 10

Sample (Ligation) 20

Phi29 Enzyme 1 5

• Aliquot 12 μl of the different dNTPs in the appropriate PCR tubes.

• Add 168 μl mastermix into each tube.

• Place the tubes in the PCR cycler.

• Add 20 μl sample the first 3 tubes and mix

• Run the “RCA30” program.

2.6.3 Dialysis S12-‐SA-‐D8

2.6.3.1 Materials


2.6.3.2 Methods




samples.


volume of the sample: 300 x 200 μl = 60 ml buffer for each sample. That makes a total of

240 ml buffer for all samples together. We can make an excess of buffer.


• 300ml . 10mM = V1 . 1000mM

à V1 = 3,0 ml 1M Tris-‐Hcl pH 8,5 + 297 ml Milli-‐Q water.

62

2.6.4 Lyophilization of S12-‐SA-‐D8

2.6.4.1 Materials

• See sections 2.4.4.1.1 and 2.4.4.2.1

2.6.4.2 Methods

• See sections 2.4.4.1.2 and 2.4.4.2.2

• The lyophilized samples are reinstated in 30 μl Hypure Molecular Biology Grade water.

• Nanodrop of the samples:

Concentration (ng/μl)

S12-‐SA-‐D8 3 nmol 118,8

S12-‐SA-‐D8 375 pmol 88,6

S12-‐SA-‐D8 93,8 pmol 89,7

Blanc 43,1

2.6.5 Gel of the different sized S12-‐SA-‐D8 particles

• See section 2.2.5

• Prepare a 1% agarose gel.

• Load 22,5 μl of the D8 samples and the blanc. Load 6 μl of the Log 2 ladder and 2 μl of the

25/100bp ladder.

1 2 3 4 5 6

Log 2 ladder D8 3 nmol D8 375 pmol D8 93,8 pmol Blanc 25/100bp ladder

2.6.6 QPCR with equal concentrations of the different sized products

2.6.6.1 Materials


2.6.6.2 Methods


• Dilute each sample to a concentration of 2,5 pg/μl for the QPCR.

63

2.6.7 Ligation of S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10

2.6.7.1 Materials


2.6.7.2 Methods


• Make 200 μl D7, 200 μl D8 and 400 μl G10 ligation.


PCR water 40,56 811,2

T4 Buffer, 10X 5 100

DTT, 500 mM 0,5 10


product, 0,5 μM

±2,10

Linker, 1 μM 1,34 26,8

T4 Enzyme 0,5 10

2.6.8 RCA of the different sized particles

2.6.8.1 Materials


2.6.8.2 Methods


2.6.8.2.1 Calculations

• dNTPs stock = 10 mM

• 30 min 3 nmol S12-‐SA-‐D8 ! !"#$!,! !!

= 10mM


PCR water 29,5 531


dNTPs, 10 mM 1 18





Sample (Staining) 5

64

• 30 min 375 pmol S12-‐SA-‐D8 !"# !!!!! !"#$

!,! !! = 1,25 mM

V1 . 10mM = 150 μl. 1,25 mM

àV1 = 18,77 μl 10mM stock + 131,23 μl Hypure Molecular Biology Grade water

• 30 min 93,8 pmol S12-‐SA-‐D8 !",! !!"!! !"#$

!,! !! = 0,3127 mM

V1 . 10mM = 150 μl. 0,3127 mM


• Dilute the dNTPs ten times and add 3 μl of the 1,0 mM solution to the PCR tubes instead

of 0,3 μl of the 10mM solution. Volumes lower than 1 μl aren’t pippetable accurately.

V1 . 10mM = 3 μl. 1,0 mM

à V1 = 0,3 μl + 2,7 μl Hypure Molecular Biology Grade water

2.6.8.2.2 Test RCA


PCR water 36 540


dNTPs, 1 mM 3

DTT, 500mM 0,5 7,5

Oligreen, 200X 0,25 3,75

Sample (Ligation) 5


2.6.8.2.3 Real RCA

Reagent Volume 1X (μl) ( For 500 μl) Volume 15X (For 500 μl)

PCR water 362,5 5437,5


dNTPs, 1 mM 30

DTT, 500mM 5 75

Sample (Ligation) 50


• Aliquot 30 μl dNTPs (3nmol/375 pmol/93,8 pmol) in the appropriate epitubes.

• Make a mastermix with PCR water, Buffer, DTT and enzyme.

• Aliquot 420 μl mastermix in each tube.

• Place the tubes into the 30 °C heatblock.

65

Add 50 μl of ligation to the appropriate epitube. Wait 1 min in between adding to the

next epitube.

• Take the first epitube out of the heatblock after 30 min and transfer it to he 65°C

heatblock for 15 min. Wait 1 min to transfer the next tube int the 65°C heatblock. Repeat

for the remaining tubes.

• Wait 1 min to take the consecutive tubes out of the heatblock after 15 min of heat

inactivation at 65°C.

2.6.9 Dialysis of the different sized particles

2.6.9.1 Materials


2.6.9.2 Methods




samples.


volume of the sample: 300 x 200 μl = 60 ml buffer for each sample. That makes a total of

240 ml buffer for all samples together. We can make an excess of buffer.


• 300ml . 10mM = V1 . 1000mM

à V1 = 3,0 ml 1M Tris-‐Hcl pH 8,5 + 297 ml Milli-‐Q water.

2.6.10 Lyophilization of the different sized particles

2.6.10.1 Materials

• See sections 2.4.4.1.1 and 2.4.4.2.1

2.6.10.2 Methods

• See sections 2.4.4.1.2 and 2.4.4.2.2

• Samples were reinstated in a tenth of the volumes after dialysis:

3 nmol dNTP Particles 85 μl Hypure Molecular Biology Grade water

375 pmol dNTP Particles 75 μl Hypure Molecular Biology Grade water

93,8 pmol dNTP Particles 81 μl Hypure Molecular Biology Grade water

66

2.6.11 Staining of the different sized particles

2.6.11.1 Materials


2.6.11.2 Methods


2.6.11.2.1 Calculation of the probe concentration

• A 1:5 molar ratio of probe is needed.

Ligation: 0,5 pmol

RCA: 0,05 pmol

0,05 pmol x 300 copies = 1,5 pmol of probe is needed for 1:10 molar ratio (standard)

à 3,0 pmol of probe is needed for 1:5 molar ratio

3 nmol dNTP Particles 3,0 pmol probe

375 pmol dNTP Particles 0,375 pmol probe

93,8 pmol dNTP Particles 0,0938 pmol probe

• A normal 50 μl reaction requires 3,0 pmol of probe. Our samples are ten times more

concentrated. This means that 30 pmol of probe is required for a 50 μl reaction.

• The samples were diluted (See section 2.6.10.2)

3 nmol dNTP Particles 85 μl 17,6 pmol probe

375 pmol dNTP Particles 75 μl 0,250 pmol probe

93,8 pmol dNTP Particles 81 μl 0,056 pmol probe

2.6.11.2.2 Preparation of the MJ Alexa Fluor and MJ Biotinylated probe solutions

2.6.11.2.2.1 Materials


Hypure Molecular

Biology Grade water



MJ Alexa Fluor 647 probe

Sequence: 5’/CCA AAA AGC

AAG GAT CCA ACT C/3’

MW: 7,710.6 g/mol


IA, USA

MJ biotinylated probe

Sequence: 5’/GGA TCC AAC

TCA ACG TCA CC/3’

MW: 6,424.4 g/mol


IA, USA

67

2.6.11.2.2.2 Methods

• Both stocks have a concentration of 100 μM.

• V1 . 100 μM = 25 μl . 17 μM

à V1= 4,25 μl stock + 20,75 μl Hypure Molecular Biology Grade water

• V1 . 100 μM = 50 μl . 2,5 μM


• V1 . 100 μM = 200 μl . 0,6 μM


• Nanodrop of the probe solutions

Calculated

concentration

Nanodrop

concentration

Actual Concentration

MJ Alexa Fluor 17 μM 96,9 ng/μl 12,6 μM

(MW: 7710,6 g/mol) 2,5 μM 12,5 ng/μl 1,6 μM

0,6 μM 2,6 ng/μl 0,34 μM

MJ Biotinylated 17 μM 70,7 ng/μl 11,0 μM

(MW: 6424,4 g/mol) 2,5 μM 7,4 ng/μl 1,15 μM

0,6 μM 1,8 ng/μl 0,28 μM

2.6.11.2.3 Calculations of the probe volumes

• MJ Alexa Fluor probe !",! !"#$!",! !!

= 1,38 μl

!,! !"#$!,! !!

= 1,56 μl

!,!" !"#$!,!" !!

= 1,65 μl

• MJ biotinylated probe !",! !"#$!!,! !!

= 1,60 μl

!,! !"#$!,!" !!

= 2,17 μl

!,!" !"#$!,!" !!

= 2,0 μl

68

2.6.11.4 Staining of the different sized particles

2.6.11.4.1 Materials


2.6.11.4.2 Methods


• Add 14 x 2 μl = 28 μl streptavidin beads to 1 well of the Greiner plate.


beads each time.

• Resuspend the beads in 700 μl TBST 10 mM MgCL2. Aliquot 50 μl TBST 10 mM MgCL2 +

beads int each well.


• Add 1 μl MgCl2 into each well.





• Incubate 30 min to remove the aggregates

• Transfer the samples to a 96-‐well white VWR plate.

69

3. Results


3.1.1 QPCR Amplification of the isolated DNA

Fig. 7. QPCR graph of the plasmid samples. Each sample was amplified during the QPCR.

The isolated plasmid DNA is double-‐stranded. In order to make templates for the production of

DeNAno particles with this plasmid DNA, a first amplification was carried out using QPCR. The

QPCR results showed that each sample is amplified (Fig. 7). In the next step, an asymmetric PCR

was performed to obtain ssDNA templates by amplifying the QPCR samples in the presence of

only the forward primer.

3.1.2 Gel of the QPCR products

Fig. 8. Gel of the QPCR samples. The 25/100bp ladder is loaded in wells 5 and 44. The

samples in wells 6-‐23 are Roberto’s samples. Wells 42 and 43 are loaded with negative

controls. Wells 24-‐41 are loaded in succession as followed: S105-‐50/ S105-‐52/ S105-‐53/

S105-‐58/ S106-‐61/ S106-‐62/ S106-‐63/ S106-‐64/ S106-‐65/ S106-‐66/ S106-‐68/ S106-‐71/

S115-‐74/ S115-‐76/ S115-‐77/ S115-‐79/ S115-‐81/ S115-‐89.

Fig. 9. 25/100bp Ladder

Every sample except for S105-‐50 showed a band at 100bp. The band of S105-‐50 was a bit lower,

caused by the fact that this sample is only 70 bp long (Fig.8).

70

3.1.3 Gel of the asymmetric products

Fig. 10. Gel of the asymmetric samples. The 25/100bp ladder is loaded in wells 5 and 44. The samples in wells 6-‐23 are Roberto’s samples. Wells 42 and 43 are loaded with

negative controls. Wells 24-‐41 are loaded in succession as followed: S105-‐50/ S105-‐52/

S105-‐53/ S105-‐58/ S106-‐61/ S106-‐62/ S106-‐63/ S106-‐64/ S106-‐65/ S106-‐66/ S106-‐68/

S106-‐71/ S115-‐74/ S115-‐76/ S115-‐77/ S115-‐79/ S115-‐81/ S115-‐89.

Fig. 11. 25/100bp Ladder

The ssDNA asymmetric samples showed the same results on the gel as the QPCR clones (Fig. 10).

3.1.4 Staining of the S105, S106 and S115 clones

The binding of the DeNAno particles to their specific target was tested during the staining. Only

the clones that specifically bound to their targets could be used for the knockoff project. The

particles were labeled with an Alexa Fluor MJ probe that absorbs light at 650 nm and

subsequently emits light at 685 nm. The resulting fluorescence was measured by the microplate

analyser and graphically represented (Fig. 12, 13 ,14).

Fig. 12. Staining of the S105 clones. S105-‐50, S105-‐52 and S105-‐58 bound distinctively to the IL-‐18 beads. Clone S105-‐53 barely bound to the IL-‐18 beads and couldn’t be differentiated from the background.

The height of the column is proportional to the number of DeNAno particles that are bound to the

beads.

S105-5

0

S105-5

2

S105-5

3

S105-5

80

10000

20000

30000

40000

Background

Sample

Fluo

resc

ence

Staining of the IL-18 S105 clones

IL-18Protein GpAb IgG

71

The S105 clones target IL-‐18. Protein G and pAb IgG were used as control beads. S105-‐50, S105-‐

52 and S105-‐58 bound distinctively to the IL-‐18 beads. Clone S105-‐53 barely bound to the IL-‐18

beads and couldn’t be differentiated from the background. In conclusion, S105-‐50 was able to

bind most efficiently of all the samples (Fig. 12).

Fig. 13. Staining of the 106 clones. All samples bound specifically to the IL-‐18 beads and not the Protein G or Rituximab beads.

IL-‐18 is also the target of the S106 clones. Protein G and Rituximab were used as control beads.

All samples bound specifically to the IL-‐18 beads and not the Protein G or Rituximab beads (Fig.

13). These clones showed the best results and were used in further experimetns to measure the

amount of particles that comes off the beads after 1 hour incubation. The individual DeNAno

clones shouldn’t be released from the beads if the cytokine isn’t present in the solution. The

DeNAno clones must remain bound to the beads in order to perform a successful quantitative

measurement of the displacement of DeNAno particles by the IL -‐18 cytokine.

Fig. 14. Staining of the S115 clones. These results show that the clones S115-‐76, S115-‐77, S115-‐79 and S115-‐81 bind to Cetuximab beads only. S115-‐74 and S115-‐89 bind to both Cetuximab and Rituximab beads.

S106-6

1

S106-6

2

S106-6

3

S106-6

4

S106-6

5

S106-6

6

S106-6

8

S106-7

1

1000

10000

100000

Background

Fluo

resc

ence


Beads Only

IL-18Protein GRituximab

Sample

S115-7

4

S115-7

6

S115-7

7

S115-7

9

S115-8

1

S115-8

9100

1000

10000

100000

Background

Sample

Fluo

resc

ence

Staining of the Cetuximab S115 clones

Protein GCetuximabRituximabpAb IgG

72

These results show that the DeNAno clones S115-‐76, S115-‐77, S115-‐79 and S115-‐81 bound to

Cetuximab beads only. S115-‐74 and S115-‐89 bound to both Cetuximab and Rituximab beads.

None of the clones bound to the Protein G or pAb IgG control beads. (Fig. 14).

3.1.5 Staining of the S106 clones

Aliquots of the sample were taken at different timepoints to determine the release of the DeNAno

particles from the beads during the staining (see Section 2.3.9). QPCR was performed with the

samples and standards with known concentrations (Fig. 15,16,17). A standard curve was made

by plotting the standards Ct values on the X axis and the concentration on the Y axis. The

concentrations of each sample were calculated by inserting the Ct’s into the function of the

standard curve.

Fig. 15. QPCR results S106 clones Total. Ct values Table 2. Staining of the S106 clones. Ct’s and concentrations of

are found in Table 2. the Total samples.

Fig. 16. QPCR results S106 clones Supernatant. Ct Table 3. Staining of the S106 clones. Ct’s and concentrations of

values are found in Table 3. the Supernatant samples.

Sample Ct Concentration (ng in 50 μl)

IL-‐18 S106-‐61 Total 7,84 0,394300359

IL-‐18 S106-‐62 Total 6,88 0,805351555

IL-‐18 S106-‐63 Total 7,78 0,412298798

IL-‐18 S106-‐64 Total 7,42 0,538916452

IL-‐18 S106-‐65 Total 7,86 0,388477205

IL-‐18 S106-‐66 Total 8,14 0,315429781

IL-‐18 S106-‐68 Total 8,39 0,261898069

IL-‐18 S106-‐71 Total 8,41 0,258030275

Sample Ct Concentration ( ng in 50 μl) IL-‐18 S106-‐61 SN 11,82 0,020415613

IL-‐18 S106-‐62 SN 10,85 0,042009893

IL-‐18 S106-‐63 SN 8,96 0,171386176

IL-‐18 S106-‐64 SN 11,35 0,028960815

IL-‐18 S106-‐65 SN 9,16 0,14769285

IL-‐18 S106-‐66 SN 7,87 0,385597958

IL-‐18 S106-‐68 SN 8,34 0,27182311

IL-‐18 S106-‐71 SN 9,07 0,157919883

73

Fig. 17. QPCR results S106 clones Resuspended beads. Table 4. Staining of the S106 clones. Ct’s and concentrations of

Ct values are found in Table 4. the Resuspended beads samples.

The % Supernatant/Total was calculated to determine the amount of the DeNAno particles that

were released from the beads during the 1 hour incubation. A loss of 5-‐10% was considered

acceptable. Only ± 5% of S106-‐61, S106-‐62 and S106-‐64 was released from the beads (Table 5).

Sample % Sup/Total

IL-‐18 S106-‐61 5,177680663

IL-‐18 S106-‐62 5,216342241

IL-‐18 S106-‐63 41,56843946

IL-‐18 S106-‐64 5,373897037

IL-‐18 S106-‐65 38,01840823

IL-‐18 S106-‐66 122,2452604

IL-‐18 S106-‐68 103,7896577

IL-‐18 S106-‐71 61,2020753

Table 5. Staining of the S106

clones. % Supernatant/Unwashed

Fig. 18. Staining of the IL-‐18 S106 Clones. 95% of the S106-‐61, S106-‐62 and

S106-‐64 clones stayed bound to the beads. The release of the other clones was too

high to use the particles in the knockoff project.

Sample Ct Concentration ( ng in 50 μl)

IL-‐18 S106-‐61 RB 7,38 0,555193925

IL-‐18 S106-‐62 RB No Ct Overloaded

IL-‐18 S106-‐63 RB 8,5 0,241320003

IL-‐18 S106-‐64 RB 7,14 0,663719057

IL-‐18 S106-‐65 RB 8,27 0,286353231

IL-‐18 S106-‐66 RB 12,02 0,017593252

IL-‐18 S106-‐68 RB 11,44 0,027085287

IL-‐18 S106-‐71 RB 10,05 0,07617577

IL-18 S106-61

IL-18 S106-62

IL-18 S106-63

IL-18 S106-64

IL-18 S106-65

IL-18 S106-66

IL-18 S106-68

IL-18 S106-710

50

100

150


% D

eNAn

o Re

leas

e

74

3.1.6 Staining of the S106 clones; Repeatability Test

The staining is repeated with the S106-‐61, S106-‐62 and S106-‐64 clones. Each clone is tested in

triplicate (A/B/C).

Fig. 19. QPCR results S106-‐61/62/64 clones Table 6. Repeatability staining of the S106 clones. Ct’s and

Total. Ct values are found in Table 6. concentrations of the Total samples.


Supernatant. Ct values are found in Table 7. concentrations of the Supernatant samples.


Resuspended Beads. Ct values are found in Table 8. concentrations of the Resuspended beads samples.


IL-‐18 S106-‐61A Total 9,86 0,374478518

IL-‐18 S106-‐61B Total 9,85 0,377044693

IL-‐18 S106-‐61C Total 9,42 0,505740291

IL-‐18 S106-‐62A Total 9,73 0,409245505

IL-‐18 S106-‐62B Total 9,58 0,453390874

IL-‐18 S106-‐62C Total 10,36 0,266152605

IL-‐18 S106-‐64A Total 10,84 0,191763691

IL-‐18 S106-‐64B Total 10,63 0,221335349

IL-‐18 S106-‐64C Total 10,3 0,277284928


IL-‐18 S106-‐61A SN 12,89 0,047288116

IL-‐18 S106-‐61B SN 13,42 0,032927446

IL-‐18 S106-‐61C SN 13,23 0,037489582

IL-‐18 S106-‐62A SN 11,92 0,091716319

IL-‐18 S106-‐62B SN 12,39 0,066534699

IL-‐18 S106-‐62C SN 12,31 0,070270912

IL-‐18 S106-‐64A SN 13,07 0,041818204

IL-‐18 S106-‐64B SN 13,1 0,040970155

IL-‐18 S106-‐64C SN 13,82 0,02505654

Sample Ct Concentration( ng in 50 μl)

IL-‐18 S106-‐61A RB 9,89 0,366884309

IL-‐18 S106-‐61B RB 10,2 0,296883117

IL-‐18 S106-‐61C RB 10,26 0,284963974

IL-‐18 S106-‐62A RB 8,5 0,947965518

IL-‐18 S106-‐62B RB 10,4 0,258980477

IL-‐18 S106-‐62C RB 10,42 0,255467218

IL-‐18 S106-‐64A RB 11,15 0,155175354

IL-‐18 S106-‐64B RB 11,16 0,154119226

IL-‐18 S106-‐64C RB 10,78 0,199784561

75

The % Supernatant/Total was calculated to determine the amount of the DeNAno particles that

were released from the beads during the 1 hour incubation. Sample S106-‐61 showed an average

release of 9,59 % while S106-‐62 and S106-‐64 had average releases of respectively 21,16 % and

16,45% (Fig. 22). Only sample S106-‐61 was used for the knockoff with the IL-‐18 cytokine.

Table 9. Repeatability staining of

the IL-‐18 S106 clones. Fig. 22. Repeatability staining of the IL-‐18 S106 clones. Sample S106-‐61

%Supernatant/Unwashed. showed an average release of 9,59% while S106-‐62 and S106-‐64 had average

releases of respectively 21,16 % and 16,45%.

Sample % Sup/Total

IL-‐18 S106-‐61A 12,62772457

IL-‐18 S106-‐61B 8,733035342

IL-‐18 S106-‐61C 7,412813051

IL-‐18 S106-‐62A 22,41107539

IL-‐18 S106-‐62B 14,67490922

IL-‐18 S106-‐62C 26,40248879

IL-‐18 S106-‐64A 21,80715455

IL-‐18 S106-‐64B 18,5104438

IL-‐18 S106-‐64C 9,0363873

S106-61

S106-62

S106-640

20

40

60

80

100

% D

eNAn

o Re

leas

e


S106-61S106-62S106-64

76

3.1.7 S106-‐61 knockoff by IL-‐18 cytokine

3.1.7.1 DeNAno S106-‐61 knockoff 1 by IL-‐18

The knockoff was carried out with a 1 μM IL -‐18 in TBST 1% BSA 10 mM MgCl2 solution. A TBST

1% BSA 10 mM MgCl2 solution without the IL-‐18 cytokine was used as a control. Aliquots of the

sample were taken at different timepoints to determine the release of the DeNAno particles from

the beads during the knockoff (see Section 2.3.14). QPCR was performed with the samples and

standards with known concentrations (Fig. 23). A standard curve was made by plotting the

standards Ct values on the X axis and the concentration on the Y axis. The concentrations of each

sample were calculated by inserting the Ct’s into the function of the standard curve. The DeNAno

S106-‐61 knockoff was performed in triplicate in order to validate the results.

Fig. 23. QPCR results DeNAno S106-‐61 knockoff 1. Ct

values are found in Table 10.

Table 10. DeNAno S106-‐61 knockoff 1. Ct’s and

concentrations of the IL-‐18 and control samples at the

different timepoints.

The sum of the concentrations of the Control 2 hr sample and the Control Pellet sample should

equal the Control Total sample. However, the Control results seemed to be off at first. The

concentration of the Control Pellet sample was lower than the concentration of the "Control 2

hour sample. This trent was apparant in the first two experiments. This suggested that there was

a strong loss of sample during the washing steps. A correction was carried out in order to

compensate for the change of the quantity of beads during the protocol. The concentration of the

Total and Supernatant (5min, 30min, 1 hr, 2hr) samples must be multiplied by a factor 2000

since there were 0,5 μl beads at the start and just 0,00025 μl left in the QPCR sample. The

concentration of the Pellet sample was corrected by multiplying by a factor 11111 since there

were 0,5 μl beads at the start and just 0,000045 μl left in the QPCR sample. The corrected results

showed that there was barely loss of sample during the protocol (Table 11). DeNAno S106-‐61

knockoff 1 showed that 81% of the bound DeNAno was released after 5 min incubation with the

IL-‐18 cytokine. 97% of DeNAno was released after 30 min.


IL-‐18 Total 10,21 0,173430081

IL-‐18 5 min 10,47 0,140754351

IL-‐18 30 min 10,24 0,169302495

IL-‐18 1hr 10,14 0,183456705

IL-‐18 2hr 10 0,205282493

IL-‐18 pellet 15,94 0,001742048

Beads No Ct /

Control Total 10,42 0,146520011

Control 5 min 15,37 0,002753091

Control 30 min 13,83 0,0094803

Control 1hr 12,36 0,030861317

Control 2 hr 12,28 0,032908694

Control Pellet 13,47 0,012657703

77

After 1 hour of incubation, each DeNAno particle was replaced by the IL-‐18 cytokine. DeNAno

couldn’t be detected on the beads after the washing steps. The Pellet sample couldn’t be

differentiated from the background. The Control results showed that only 1,8 % and 6,4% of

DeNAno was released from the beads after respectively 5 min and 30 min. The release increased

to 21% after 1 hour but stayed at 22% after a total of 2 hours of incubation. The Control pellet

showed that there was still 48% of DeNAno bound to the beads after the incubation and the

washing steps. (Table 11).

Table 11. DeNAno S106-‐61 knockoff 1. Corrected concentrations of the samples.


The second DeNAno knockoff by IL-‐18 showed that 77% of the bound DeNAno was released after

5 min incubation with the IL-‐18 cytokine. After 30 min of incubation, each DeNAno particle was

replaced by the IL-‐18 cytokine. DeNAno couldn’t be detected on the beads after the washing

steps. The Pellet sample couldn’t be differentiated from the background. The Control results

showed that just 1 % of DeNAno was released from the beads after 5 min. The release increased

barely to 3,7% after 30 min and 8,5% after 1 hour. There was a total release of 14,7% after 2

hours of incubation. The Control pellet showed that there was still 49% of DeNAno bound to the

beads after the incubation and the washing steps (Table 13.).

Sample Corrected amount RCA (ng) Percentage

IL-‐18 Total 346,8601629 100

IL-‐18 5 min 281,5087011 81,15913307

IL-‐18 30 min 338,6049903 97,62002863

IL-‐18 1hr 366,9134106 105,7813638

IL-‐18 2hr 410,5649859 118,36614

IL-‐18 pellet 19,35589857 5,580317559

Control Total 293,0400222 100

Control 5 min 5,506181243 1,878986086

Control 30 min 18,96059931 6,470310495

Control 1hr 61,72263476 21,06286858

Control 2 hr 65,81738774 22,46020433

Control Pellet 140,6397365 47,99335445

78

Fig. 24. QPCR results DeNAno S106-‐61 knockoff 2. Ct


Table 12. DeNAno S106-‐61 knockoff 2. Ct’s and

concentrations of the IL-‐18 and control samples at the

different timepoints.

Table 13. DeNAno S106-‐61 knockoff 2. Corrected concentrations of the samples.


Additional aliquots were taken to determine if there was a loss of sample during the washing

steps. The Ct of the IL-‐18 total sample was too high. As a result, the samples showed release

percentages that were greater than 100%. The results showed that 110% of DeNAno was

released after 5 min. The release increased to a maximum of 160% after 2 hours of incubation.

DeNAno couldn’t be detected on the beads after the washing steps. The Pellet sample couldn’t be

differentiated from the background (Table 15.).

Sample Ct Concentration ( ng in 50 μl) IL-‐18 Total 9,56 0,215460805

IL-‐18 5 min 9,88 0,16755055

IL-‐18 30 min 9,48 0,22944251

IL-‐18 1hr 9,58 0,21210059

IL-‐18 2hr 9,52 0,222341782

IL-‐18 pellet 15,28 0,00240446

Beads No Ct /

Control Total 9,34 0,256128965

Control 5 min 15,12 0,002726646

Control 30 min 13,51 0,009663935

Control 1hr 12,47 0,021884009

Control 2 hr 11,78 0,037638969


Sample Corrected Amount RCA (ng) Percentage

IL-‐18 Total 430,9216109 100

IL-‐18 5 min 335,1010992 77,76381846

IL-‐18 30 min 458,8850195 106,4892101

IL-‐18 1hr 424,201181 98,44045188

IL-‐18 2hr 444,6835637 103,1936093

IL-‐18 pellet 26,71595041 6,199723971


Control 5 min 5,453291183 1,064559642

Control 30 min 19,32787018 3,773073886

Control 1hr 43,76801717 8,544136583

Control 2 hr 75,27793899 14,69531941

Control Pellet 250,9185833 48,98285976

79

Fig. 25. QPCR results DeNAno S106-‐61

knockoff 3. Ct values are found in Table 14.

Table 14. DeNAno S106-‐61 knockoff 3. Ct’s and concentrations of the

IL-‐18 and control samples at the different timepoints.

Table 15. DeNAno S106-‐61 knockoff 3. Corrected concentrations of the samples

and percentages of release.

Sample Ct Concentration ( ng in 50 μl) IL-‐18 Total 8,55 0,635124767

IL-‐18 5 min 8,4 0,702686594

IL-‐18 30 min 8,5 0,656890914

IL-‐18 1hr 7,92 0,971068913

IL-‐18 2hr 7,78 1,067150886

IL-‐18 Resuspended 1 13,55 0,021849344

IL-‐18 Resuspended 2 13,9 0,017258337

IL-‐18 Resuspended 3 13,83 0,018092011

IL-‐18 Pellet 14,45 0,011913042

IL-‐18 Beads No Ct /

Control Total 7,73 1,103722856

Control 5 min 12,53 0,043448642

Control 30 min 11,3 0,099535428

Control 1hr 10,59 0,160614009

Control 2 hr 9,52 0,330335647

Control Resuspended 1 11,49 0,087572491




Sample Corrected Amount RCA (ng) Percentage

IL-‐18 Total 1270,249534 100

IL-‐18 5 min 1405,373188 110,6375677

IL-‐18 30 min 1313,781829 103,4270664

IL-‐18 1hr 1942,137825 152,894197

IL-‐18 2hr 2134,301772 168,0222441

IL-‐18 Resuspended 1 242,7680565 19,11183984

IL-‐18 Resuspended 2 196,6587515 15,48189913

IL-‐18 Resuspended 3 211,4594217 16,64707729

IL-‐18 Pellet 142,8135486 11,24295225


Control 5 min 86,8972841 3,936553622

Control 30 min 199,0708561 9,01815411

Control 1hr 321,2280173 14,55202344

Control 2 hr 660,671294 29,92922048




Control Pellet 823,6603324 37,31282397

80

The Control results showed that just 3,9% of DeNAno was released from the beads after 5 min.

The release increased barely to 9% after 30 min and 14,6% after 1 hour. There was a total

release of 29,9% after 2 hours of incubation. The three Control Resuspended samples showed

that there was little loss of sample during the washing steps. There was still 37% of DeNAno

bound to the beads after the incubation and the washing steps. (Table 15).

3.1.7.4 DeNAno S106-‐61 knockoff merged results

The results of the three S106-‐61 knockoffs by IL-‐18 were merged into one graph to determine

the timepoints for the titration. The mean release of DeNAno from the beads was 89,5% after 5

min, 102,5% after 30 min, 119% after 1 hour and 129% after two hours of incubation with IL-‐18

was calculated. The Control samples showed a mean release of 2,29% after 5 min, 6,4% after

30min, 14,7% afer 1 hour and 22,4% after two hours of incubation (Fig. 26).

Fig. 26. DeNAno S106-‐61 knockoff by the IL-‐18 cytokine. The mean release of DeNAno from the beads was 89,5% after 5 min, 102,5% after 30 min, 119% after 1 hour and 129% after two hours of incubation with IL-‐18.

3.1.8 S106-‐61 titration with different concentrations of IL-‐18

The titration was performed by incubating the S106-‐61 clone with different concentrations of the

IL -‐18 in TBST 1% BSA 10 mM MgCl2 solution. Aliquots of the samples were taken at the 5 min

and the 1 hour timepoints. QPCR was performed with the samples and standards with known

concentrations. A standard curve was made by plotting the standards Ct values on the X axis and

the concentration on the Y axis. The concentrations of each sample were calculated by inserting

the Ct’s into the function of the standard curve. Based on the concentrations we were able to

calculate the percentage of DeNAno that was released from the beads after incubation with the

different concentrations of the IL-‐18 cytokine.

SN 5min

SN 30 m

in

SN 1 hr

SN 2 hr

Pellet

0

50

100

150

200

Knockoff S106-61

Sample

%

IL-18 Control

81

3.1.8.1 S106-‐61 titration 1 with IL-‐18

By equalizing the Total sample to 100%, we could determine the percentages of DeNAno that

came off the beads at the 5 min and 1 hour timepoints. The values may differ from the expected

pattern because the Ct values were close to each other at higher concentrations of the IL-‐18

cytokine.

Fig. 27. QPCR results S106-‐61 titration 1

with IL-‐18 5 min. Ct values are found in Table

16.

Table 16. S106-‐61 titration 1 with IL-‐18 5 min. Ct’s and concentrations

of the IL-‐18 and control samples.

Fig. 28. QPCR results S106-‐61 titration 1

with IL-‐18 1 hr. Ct values are found in Table

17.

Table 17. S106-‐61 titration 1 with IL-‐18 1 hr. Ct’s and concentrations

of the IL-‐18 and control samples.

At the 5 min timepoint, there was a release of 1,8% of DeNAno at the lowest concentration of the

cytokine. The release increased barely to 3,8% at the 3,91 nM concentration. A shift could be

observed at the 7,81 nM concentration. 16% of DeNAno was released at 7,81 nM which increased

to 25,7% at the 15,63 nM concentration and 47,4% at the 31,25 nM concentration. At the

maximum concentration of 1 μM there was a total release of 77,4% of DeNAno. The control or

background showed a release of 1,9% of DeNAno after 5 min of incubation (Table 18).

Sample Ct Concentration ( ng in 50 μl) IL-‐18 Sup 5min 0,98 nM 12,91 0,013853277

IL-‐18 Sup 5min 1,95 nM 12,53 0,017883085

IL-‐18 Sup 5min 3,91 nM 11,73 0,030612229

IL-‐18 Sup 5min 7,81 nM 10,3 0,080018666

IL-‐18 Sup 5min 15,63 nM 8,81 0,21776927

IL-‐18 Sup 5min 31,25 nM 8,37 0,292681636

IL-‐18 Sup 5min 62,5 nM 7,65 0,474791275

IL-‐18 Sup 5min 125 nM 7,62 0,484459189

IL-‐18 Sup 5min 250 nM 7,72 0,452976462

IL-‐18 Sup 5min 1 µM 7,42 0,554141052

IL-‐18 Sup 5min control 12,9 0,013946674

Sample Ct Concentration ( ng in 50 μl) IL-‐18 Sup 1 hr 0,98 nM 10,16 0,08791146

IL-‐18 Sup 1 hr 1,95 nM 9,61 0,12721669

IL-‐18 Sup 1 hr 3,91 nM 8,34 0,298641352

IL-‐18 Sup 1 hr 7,81 nM 7,67 0,468453423

IL-‐18 Sup 1 hr 15,63 nM 7,23 0,629600834

IL-‐18 Sup 1 hr 31,25 nM 6,93 0,770211474

IL-‐18 Sup 1 hr 62,5 nM 7,5 0,525139969

IL-‐18 Sup 1 hr 125 nM 7,53 0,514660224

IL-‐18 Sup 1 hr 250 nM 7,42 0,554141052

IL-‐18 Sup 1 hr 1 µM 7,09 0,691702719

IL-‐18 Sup 1 hr control 10,82 0,056421831

82

At the 1 hour timepoint, there was a release of 11,6% of DeNAno at the lowest concentration of

the cytokine. A shift could be observed at the 3,91 nM concentration, where the release increased

to 37,2%. At a concentration of 7,81 nM, there was already a release of 94%. At the maximum

concentration of 1 μM there was a total release of 96,7% of DeNAno. The control or background

showed a release of 7,7% of DeNAno after 1 hour of incubation (Table 18).

Table 18. S106-‐61 titration 1 with IL-‐18 . Percentages of

release at the 5 min and 1 hour timepoints.



cytokine. The release increased barely to 4% at the 3,91 nM concentration. A shift could be



maximum concentration of 1 μM there was a total release of 66% of DeNAno. The control or


Fig. 29. QPCR result S106-‐61 titration 2 with

IL-‐18 5 min. Ct values are found in Table 19.


of the IL-‐18 and Control samples.

Percentages

Total Sup 5 min Sup 1 hr

0,98 nM 100 1,835257424 11,64635376

1,95 nM 100 2,499954936 17,7841792

3,91 nM 100 3,817473478 37,24183065

7,81 nM 100 16,07909055 94,13184979

15,63 nM 100 25,73548892 74,40482883

31,25 nM 100 47,43344289 124,8243055

62,5 nM 100 65,92866901 72,91999882

125 nM 100 58,81195967 62,47827892

250 nM 100 51,41650167 62,899503

1 µM 100 77,46580884 96,69615793

Control 100 1,91075906 7,730052474


IL-‐18 Sup 5min 0,98 nM 12,32 0,017820908

IL-‐18 Sup 5min 1,95 nM 11,39 0,033538928

IL-‐18 Sup 5min 3,91 nM 11,08 0,041408449

IL-‐18 Sup 5min 7,81 nM 9,45 0,125432784

IL-‐18 Sup 5min 15,63 nM 8 0,336187081

IL-‐18 Sup 5min 31,25 nM 7,66 0,423622963

IL-‐18 Sup 5min 62,5 nM 7,49 0,475530887

IL-‐18 Sup 5min 125 nM 7,37 0,515956972

IL-‐18 Sup 5min 250 nM 7,4 0,505539154

IL-‐18 Sup 5min 1 µM 7,41 0,502113503


83

Fig. 30. QPCR result S106-‐61 titration 2 with IL-‐

18 1 hr. Ct values are found in Table 20.

Table 20. S106-‐61 titration 2 with IL-‐18 1 hr. Ct’s and

concentrations of the IL-‐18 and Control samples.

At the 1 hour timepoint, there was a release of 9,7% of DeNAno at the lowest concentration of the

cytokine. The release increased to 26,9% at the 3,91 nM concentration. A shift could be observed

at the 7,81 nM concentration, where the release increased to 94,7%. At the maximum

concentration of 1 μM there was a total release of 70% of DeNAno. The control or background


Percentage


0,98 nM 100 2,102591552 9,708599306

1,95 nM 100 4,852450983 20,37146543

3,91 nM 100 4,011253059 26,9209895

7,81 nM 100 23,49811869 94,70585512

15,63 nM 100 66,95439956 102,7570399

31,25 nM 100 69,7423372 79,90140625

62,5 nM 100 78,82221417 78,82221417

125 nM 100 74,1434113 76,70736161

250 nM 100 79,35997578 82,66447355

1 µM 100 66,05007683 70,21815169

Control 100 2,312573065 8,767244799

Table 21. S106-‐61 titration 2 with IL-‐18. Percentages of







maximum concentration of 1 μM there was a total release of 82% of DeNAno. The control or


Sample Ct Concentration ( ng in 50 μl )

IL-‐18 Sup 1 hr 0,98 nM 10,07 0,08228705

IL-‐18 Sup 1 hr 1,95 nM 9,28 0,140802478

IL-‐18 Sup 1 hr 3,91 nM 8,28 0,277907281

IL-‐18 Sup 1 hr 7,81 nM 7,4 0,505539154

IL-‐18 Sup 1 hr 15,63 nM 7,37 0,515956972

IL-‐18 Sup 1 hr 31,25 nM 7,46 0,485330315

IL-‐18 Sup 1 hr 62,5 nM 7,49 0,475530887

IL-‐18 Sup 1 hr 125 nM 7,32 0,53379926

IL-‐18 Sup 1 hr 250 nM 7,34 0,526589475

IL-‐18 Sup 1 hr 1 µM 7,32 0,53379926

IL-‐18 Sup 1 hr control 10,75 0,051824431

84

Fig. 31. S106-‐61 titration 3 with IL-‐18 5 min. Ct values are found in Table 22.

Table 22. S106-‐61 titration 3 with IL-‐18 5 min. Ct’s and

concentrations of the IL-‐18 and Control samples.

At the 1 hour timepoint, there was a release of 18,9% of DeNAno at the lowest concentration of

the cytokine. A shift could be observed at the 3,91 nM concentration, where the release increased

to 57,2%. At a concentration of 7,81 nM, there was already a release of 84,5%. At the maximum

concentration of 1 μM there was a total release of 92,8% of DeNAno. The control or background

shows a release of 7,8% of DeNAno after 1 hour of incubation (Table 24).

Fig. 32. IL-‐18 Titration 3 S106-‐61 1 hr. Ct


Table 23. IL-‐18 Titration 3 S106-‐61 1 hr. Ct’s and concentrations of

the IL-‐18 and Control samples.


IL-‐18 Sup 5min 0,98 nM 14,51 0,016724359

IL-‐18 Sup 5min 1,95 nM 13,84 0,026234086

IL-‐18 Sup 5min 3,91 nM 13,17 0,041151192

IL-‐18 Sup 5min 7,81 nM 11,25 0,149509376

IL-‐18 Sup 5min 15,63 nM 10,06 0,332605197

IL-‐18 Sup 5min 31,25 nM 9,84 0,385592405

IL-‐18 Sup 5min 62,5 nM 9,74 0,412391812

IL-‐18 Sup 5min 125 nM 9,6 0,453068869

IL-‐18 Sup 5min 250 nM 9,59 0,456123427

IL-‐18 Sup 5min 1 µM 9,58 0,459198578



IL-‐18 Sup 1 hr 1,95 nM 11,4 0,135174909

IL-‐18 Sup 1 hr 3,91 nM 9,96 0,355721893

IL-‐18 Sup 1 hr 7,81 nM 9,5 0,484558019

IL-‐18 Sup 1 hr 15,63 nM 9,35 0,535942417

IL-‐18 Sup 1 hr 31,25 nM 9,41 0,514765215

IL-‐18 Sup 1 hr 62,5 nM 9,76 0,406886913

IL-‐18 Sup 1 hr 125 nM 9,42 0,511317946

IL-‐18 Sup 1 hr 250 nM 9,4 0,518235726

IL-‐18 Sup 1 hr 1 µM 9,41 0,514765215

IL-‐18 Sup 1 hr control 12,87 0,050341611

85






observed at the 15,63 nM concentration. 26,9% of DeNAno was released at 15,63 nM which

increased to 60,8% at the 31,25 nM concentration. At the maximum concentration of 1 μM there

was a total release of 69,8% of DeNAno. The control or background showed a release of 0,7% of

DeNAno after 5 min of incubation (Table 27).

Fig. 33. S106-‐61 titration 4 with IL-‐18 5 min.

Ct values are found in Table 25.



At the 1 hour timepoint, there was a release of 6,6% of DeNAno at the lowest concentration of the

cytokine. A shift could be observed at the 3,91 nM concentration, where the release increased to

21,9%. At a concentration of 7,81 nM, there was already a release of 73,3%. At the maximum

concentration of 1 μM there was a total release of 80% of DeNAno. The control or background


Percentage


0,98 nM 100 3,270833469 18,89283939

1,95 nM 100 4,765134758 24,55304327

3,91 nM 100 6,623153899 57,25231149

7,81 nM 100 26,08367234 84,536856

15,63 nM 100 58,41810896 94,13184979

31,25 nM 100 75,41147485 100,6741928

62,5 nM 100 76,43174011 75,41147485

125 nM 100 112,8565614 127,3660345

250 nM 100 80,11260272 91,02188213

1 µM 100 82,84983027 92,87531088

Control 100 1,962811314 7,834634753


IL-‐18 Sup 5min 0,98 nM 13,99 0,024056852

IL-‐18 Sup 5min 1,95 nM 14,81 0,013662823

IL-‐18 Sup 5min 3,91 nM 13,3 0,038724423

IL-‐18 Sup 5min 7,81 nM 12,52 0,066328169

IL-‐18 Sup 5min 15,63 nM 10,8 0,217302768

IL-‐18 Sup 5min 31,25 nM 9,71 0,460961093

IL-‐18 Sup 5min 62,5 nM 9,46 0,547737281

IL-‐18 Sup 5min 125 nM 9,44 0,555347657

IL-‐18 Sup 5min 250 nM 9,56 0,511221475

IL-‐18 Sup 5min 1 µM 9,48 0,540231196


86

Fig. 34. S106-‐61 titration 4 with IL-‐18 1 hr.


Table 26. S106-‐61 titration 4 with IL-‐18 1 hr. Ct’s and concentrations




3.1.8.5 Merged results of the S106-‐61 titration with IL-‐18

The results of the IL-‐18 Titrations were combined into two graphs to determine the

concentration of IL-‐18 that was necessary to release 50% of the DeNAno particles from the

beads. Furthermore, we also wanted to determine the concentration at which we could

distinguish the release from the background, in other words, the sensitivity of the titration. The 5

min titration showed a mean release of 2,75% at 0,98 nM, 3,5% at 1,95 nM, 4,8% at 3,91 nM,

18,8% at 7,81nM, 44,5% at 15,63 nM, 63,4% at 31,25 nM, 71% at 62,5 nM, 80,6% at 125 nM,

71,3% at 250 nM and 74% at 1 μM. The background showed a mean release of 1,7%. The 1 hour

titration showed a mean release of 11,71% at 0,98 nM, 17,42% at 1,95 nM, 35,8% at 3,91 nM,

86,7% at 7,81nM, 86,2% at 15,63 nM, 96,6% at 31,25 nM, 72,9% at 62,5 nM, 85,0% at 125 nM,

78,5% at 250 nM and 85,1% at 1 μM. The background showed a mean release of 6,9% (Fig. 35).


IL-‐18 Sup 1 hr 1,95 nM 12,9 0,051031437

IL-‐18 Sup 1 hr 3,91 nM 11,13 0,173055999

IL-‐18 Sup 1 hr 7,81 nM 9,56 0,511221475

IL-‐18 Sup 1 hr 15,63 nM 9,34 0,595015332

IL-‐18 Sup 1 hr 31,25 nM 9,29 0,615899373

IL-‐18 Sup 1 hr 62,5 nM 9,42 0,563063773

IL-‐18 Sup 1 hr 125 nM 9,5 0,532827973

IL-‐18 Sup 1 hr 250 nM 9,5 0,532827973

IL-‐18 Sup 1 hr 1 µM 9,27 0,624456808

IL-‐18 Sup 1 hr control 13,54 0,032815063

Percentages


0,98 nM 100 3,799651286 6,598564082

1,95 nM 100 1,866906876 6,973005512

3,91 nM 100 4,904645539 21,91842437

7,81 nM 100 9,511619388 73,31039256

15,63 nM 100 26,95870137 73,81793063

31,25 nM 100 60,85060101 81,30370997

62,5 nM 100 62,98635523 64,74880582

125 nM 100 76,93780529 73,81793063

250 nM 100 74,32898246 77,47045645

1 µM 100 69,85400267 80,74470313

Control 100 0,730504624 3,402535002

87

Fig. 35. S106-‐61 titration with IL-‐18. The 5 min titration plateaus at 62,5 nM while the 1 hr titration plateaus at 7,81 nM. In the 5 min titration, the release of DeNAno couldn’t be differentiated from the background at the lower concentrations. This

lowers the sensitivity of the test.

3.2 Lyophilization

3.2.1 Lyophilization of DeNAno

Samples S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10 were tested in a staining after the lyophilization.

The lyophilized and redissolved samples were compared to the dialysed control samples. QPCR

was performed with the samples and standards with known concentrations (Fig. 36,37). A

standard curve was made by plotting the standards Ct values on the X axis and the concentration

on the Y axis. The concentrations of each sample were calculated by inserting the Ct’s into the

function of the standard curve.

Fig. 36. QPCR Result staining lyophilized samples. Table 28. Ct’s and concentration of the lyophilized

Ct values are found in Table 28. samples.


Lyo D7 11,78 0,278011185

Lyo D8 11,25 0,400107633

Lyo G10 21,6 0,00032694

Lyo G10 Bio 13,48 0,086477342

88

Fig. 37. QPCR Result staining Control samples. Table 29. Ct’s and concentration of the Control samples.


These results showed that the lyophilized samples could still bind to the beads. S12-‐SA-‐D8 bound

the beads better than S12-‐SA-‐D7. S13-‐G10 was only able to bind to the streptavidin beads if it

was labeled with the Biotin probe and was used as a negative control (Fig. 38).

Fig. 38. Lyophilization of DeNAno. The lyophilized samples could still bind to the beads. S12-‐SA-‐D8 bound the beads better

than S12-‐SA-‐D7. S13-‐G10 was only able to bind to the streptavidin beads if it was labeled with the Biotin probe and was used

as a negative control.

3.2.2 Applications of lyophilization

3.2.2.1 Lyophilization in vials versus lyophilization in epitubes

Samples S12-‐SA-‐D7 and S12-‐SA-‐D8 were dialysed and split in equal parts into the lyophilization

vials and 1,5 ml epitubes with screwcaps. The samples were reinstated in the same volume of

Hypure Molecular Biology Grade water. Nanodrop was performed to compare the difference in

concentration between the two samples (Table 29).

Lyo S

12-S

A-D7

Dia S12

-SA-D

7

Lyo S

12-S

A-D8

Dia S12

-SA-D

8

Lyo S

13-G

10

Dia S13

-G10

Lyo S

13-G

10 B

io

Dia S13

-G10

Bio

0.0

0.2

0.4

0.6

Lyophilization of DeNAno

Con

cent

ratio

n (n

g)

Lyo S12-SA-D7Dia S12-SA-D7Lyo S12-SA-D8Dia S12-SA-D8Lyo S13-G10Dia S13-G10Lyo S13-G10 BioDia S13-G10 Bio


Dia D7 11,46 0,34635995

Dia D8 10,81 0,541304654

Dia G10 23,11 0,000115875

Dia G10 Bio 12,09 0,224688147

89

Table 29. Lyophilization in vials versus lyophilization in epitubes.

Fig. 39. Lyophilization in vials versus lyophilization in epitubes. There is a 16% loss of sample S12-‐SA-‐D7 and a 10% loss

of sample S12-‐SA-‐D8.

There was a 16% loss of S12-‐SA-‐D7 sample between the vials and the epitubes. S12-‐SA-‐D8

showed a loss of 10% between the vials and the epitubes (Fig. 39).

3.2.2.2 Long term storage of lyophilized samples

Samples S12-‐SA-‐D7 and S12-‐SA-‐D8 were lyophilized and stored at -‐20°C. A staining was

performed to test if the samples were still able to bind the beads after one month of incubation.

Fig. 40. Staining S12-‐SA-‐D7 and S12-‐SA-‐D8 after long term storage. Both samples are still able to bind to the

streptavidin beads.

S12-S

A-D7

S12-S

A-D8

0

20

40

60

80

100

Lyophilization,in,vials,versus,lyophilization,in,epitubes

Con

cent

ratio

n (n

g/µl

) VialsEpitubes

0

10000

20000

30000

40000

50000

Background

Fluo

resc

ence

Staining Long Term Storage

S12-SA-D7S12-SA-D8Beads Only

Vials Epitubes Difference

S12-‐SA-‐D7 75,0 ng/ μl 63,0 ng/ μl 16%

S12-‐SA-‐D8 81,0 ng/ μl 72,6 ng/ μl 10%

90

Both samples were able to bind the beads and could be differentiated from the background on

the microplate analysis (Fig. 40).

3.2.2.3 Lyophilization of different sized particles

In the first part of the applications of lyophilization, different sized particles of S12-‐SA-‐D7 and

S12-‐SA-‐D8 were made. QPCR was performed with equal concentrations of each sample. Each

sample should have the same Ct as a result of the same concentrations. This was not the case. A

gel was made to check the sizes of the different particles (Fig. 41). The different sized particles

weren’t able to be visualised on the gel.

Fig. 41. Gel different sized S12-‐SA-‐D7 and S12-‐SA-‐D8. The log 2 ladder (Fig. 43) was loaded in well 1 while the 25/100bp

ladder (Fig.42)was loaded in well 8. Wells 2,3 and 4 respectively contained the 30min 3 nmol D7, 30 min 93,75 pmol D7 and

the 5 min 93,75 pmol D7 samples.. Wells 5,6 and 7 respectively contain the 30min 3 nmol D8, 30 min 93,75 pmol D8 and the 5

min 93,75 pmol D8 samples. The different sized particles weren’t able to be visualised on the gel.

Fig. 42. 25/100 bp ladder18. Fig. 43. Log 2 Ladder19.

91

3.2.2.3.1 Different Sized Particles S12-‐SA-‐D8

The protocol was altered to attempt to visualize the different sized particles on a gel. Only sample

S12-‐SA-‐D8 was tested. The different sized particles were formed by using a 3 nmol, 375 pmol and

93,8 pmol dNTP concentration during the rolling circle amplification.

3.2.2.3.1.1 Gel Different Sized Particles S12-‐SA-‐D8

There was RCA in well 2,3 and 4. There was no sample in the blanc well 5. Each well showed a

band at 100bp and 200bp. We suggest that these are the circular templates that were still in the

solutions. The DeNAno particles were too big to move through the gelpores and stayed in the top

of the well (Fig. 44).

Fig. 44. Gel Different Sized Particles S12-‐SA-‐D8. The log 2 ladder was loaded in well 1 while the 25/100bp ladder was

loaded in well 6. Well 2,3,4 and 5 respectively contained the 3nmol dNTP S12-‐SA-‐D8, 375 pmol S12-‐SA-‐D8, 93,8 pmol S12-‐SA-‐

D8 and blanc samples.

Fig. 45. 25/100 bp ladder18. Fig. 46. Log 2 Ladder19.

92

3.2.2.3.1.2 QPCR with equal concentrations

Each sample was diluted to a concentration of 2,5 pg/μl. Sample 3nmol dNTP D8 came up first,

followed by 375 pmol dNTP D8 and 93,8 pmol dNTP D8 (Fig. 45).

Fig. 45. QPCR results different sized S12-‐SA-‐D8 particles. Sample 3nmol dNTP D8 came up first, followed by 375 pmol

dNTP D8 and 93,8 pmol dNTP D8.

3.2.2.3.2 Different Sized Particles S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10

Samples S12-‐SA-‐D7, S12-‐SA-‐D8 and S13-‐G10 were tested in a staining after the lyophilization.

S13-‐G10 was used as a negative control, while S13-‐G10 Bio was used as a positive control. Each

sample showed the same trend. Staining of S12-‐SA-‐D7 and S12-‐SA-‐D8 was still possible when

RCA was performed with a 375 pmol dNTP concentration. The smallest particles were difficult to

differentiate from the background (Fig. 46).

Fig. 46. Staining of S12-‐SA-‐D7, S12-‐SA-‐D8, S13-‐G10 and S13-‐G10 Bio. Three different sized particles were formed

for each sample by adjusting the dNTP concentration during the rolling circle amplification. S13-‐G10 was the

negative control; S13-‐G10 Bio was the positive control. Staining of D7 and D8 was still possible when RCA was

performed with a 375 pmol dNTP concentration. The smallest particles were difficult to differentiate from the

background.

S12-S

A-D7

S12-S

A-D8

S13-G

10

S13-G

10 B

io100

1000

10000

100000

Sample

FLuo

resc

ence

Staining of the different sized particles

3nmol375 pmol93,8 pmol

93

4. Discussion


Our project started with 36 different plasmid. In order to be able to investigate all the clones,

they were divided between two interns, Roberto Monterosso and me. The plasmid DNA was

isolated from E.Coli. Each plasmid contained a different 100bp insert. The isolated plasmid DNA

was amplified with QPCR. The QPCR results showed that each clone was amplificated. Only the

SsDNA templates were needed for the production of the DeNAno particles. These ssDNA template

strands were produced by amplifying the QPCR products by asymmetric PCR. The QPCR and

asymmetric samples were investigated with gelelectrophoresis. The gel results showed that

every sample except for S105-‐50 had a band at 100bp. The band of S105-‐50 was a bit lower since

this template is only 70 bp long. DeNAno particles were produced through rolling circle

amplification after making the ssDNA templates circular by ligation. Protein G beads, coated with

the target antibodies, were incubated with the DeNAno particles during the staining. All IL-‐18

clones except sample S105-‐53 were able to bind the IL-‐18 beads. The clones S115-‐76, S115-‐77,

S115-‐79 and S115-‐81 bound to the Cetuximab beads only while clones S115-‐74 and S115-‐89

bound to both Cetuximab and Rituximab beads. These two clones are called “sticky”, meaning

that they don’t specifically bind to the Cetuximab antibody, and can’t be used in a knockoff. The

IL-‐18 S106 clones showed the best results of all the clones and were tested in further stainings.

The samples were incubated for an extra hour after the washing steps. Aliquots of the sample

were taken before the incubation (Total), after one hour of icubation (Supernatant) and after the

washing steps (Resuspended beads) to determine the release of the DeNAno particles from the

beads during the staining. The clones that showed the following optimal results were selected:

1. The Ct of the "Total “ sample came up first.

2. The Ct of the “Resuspended beads” sample was close to the Ct of the

“Total” sample.

3. The Ct of the “Supernatant” sample was a couple of Ct’s lower than the

Ct of the “Total” sample.

These results suggested that DeNAno stayed on the beads during the 1 hour incubation and the

following washing steps. 95% of the samples S106-‐61, S106-‐62 and S106-‐64 stayed on the beads.

The staining was repeated in triplet with these three samples to validate the results. Only sample

S106-‐61 showed acceptable results for the knockoff. We wanted to determine the timepoints for

the titration during the knockoff. The clones were incubated with a high concentration of the

cytokine. The amount of DeNAno that was released in the control samples determined the height

of the background. The release of the ”2 hour“ Control sample was too high to use in the titration.

Another factor to determine the timepoints was the difference between the release of DeNAno,

caused by the IL-‐18 cytokine at the high concentration, and the Control particles that just

released the beads during incubation. The “5 min” knockoff showed the biggest difference

between these two values. The problem is that not every IL-‐18 cytokine will be able to displace a

94

DeNAno particle in those 5 minutes at the lower concentrations of the cytokine. In the “1 hour”

knockoff, each IL -‐18 molecule was able to interact with an IL -‐18 antibody, while keeping the

background low enough. Both titrations were performed and compared to eachother. The results

showed the range of the titration after 5 min and 1 hour incubation. The “5 min” titration

plateaus at a 62,5 nM concentration, while the “1 hour” titration already starts reaching a plateau

at the lower 7,81 nM concentration. The release of DeNAno at the lower concentrations of the

cytokine is negligible after 5 min. This lowers the sensitivity of the test. We can differentiate the

release of DeNAno from the background at a 1 nM concentration of the IL-‐18 cytokine. IL-‐18 has

plasma levels of around 100 pg/ml. This equaled to a concentration of 5,55 pM. The sensitivity of

the titration was therefore almost a 200 fold to low to detect the IL-‐18 in human blood samples.

4.2 Lyophilization

4.2.1 Lyophilization of DeNAno particles

Lyophilization is a method of removing water from frozen material. A DeNAno solution is frozen

with liquid nitrogen at −195.79 °C. The frozen solution is lyophilized to remove the present water

so that only the solutes remain as solids. This powder has various applications. First of all, it is a

easy way to store DeNAno long term in de freezer or transport it. Furthermore, it is possible to

redissolve the solids in a smaller volume of water to obtain a higher concentrated solution.

Lyophilization of the S12-‐SA-‐D7, D8-‐SA-‐S12 and S13-‐G10 samples was attempted in these

experiments. A staining was performed in order to verify that the DeNAno particles were intact

and were still able to bind to the streptavidin beads. S13-‐G10 was only able to bind to the

streptavidin beads if it was labeled with the Biotin probe and was used as a negative control. The

results showed that the S12-‐SA-‐D7 and S12-‐SA-‐D8 samples were able to bind after lyophilisation.

The amount of particles that could bind after lyophilization is lower than the amount of Control

samples that bound, and this in both samples.

4.2.2 Applications for DeNAno particles

After it became clear that it was possible to lyophilize DeNAno particles, different hypotheses

were tested to determine the limits of lyophilization. First of all, lyophilization was performed

with glass vials. These vials didn’t have a proper way to be sealed and were difficult to use. We

wanted to test the possibility to lyophilize in epitubes that could be hermetically sealed with a

screw cap. The results showed that there is a 16% loss of SA-‐S12-‐D7 and 10% loss of SA-‐S12-‐D8

between the glass vials and the epitubes. This is an acceptable result considering the fact that the

epitubes are far easier to use. In a second experiment, we tried to store the lyophilized DeNAno

particles during a long period of time at -‐20°C. The lyophilized SA-‐S12-‐D7 and SA-‐S12-‐D8 were

stored in the glass vials, sealed with a plastic plug and wrapped with Parafilm to prevent contact

with the surrounding environment. The particles were tested after one month in a staining to

check if they were still able to bind the streptavidin beads. The result showed that both S12 and

S12-‐SA-‐D7-‐D8-‐SA will still be able to bind to the streptavidin beads. The last part of the project

95

was an attempt to stain different sized particles. In the first attempt we weren’t able to visualize

the particles on the gel. The RCA protocol was altered in order to make slightly bigger particles.

This protocol was first tested with only sample S12-‐SA-‐D8. The results showed that different

sized particles were formed. The protocol was then performed with samples S12-‐SA-‐D7, S12-‐SA-‐

D8, S13-‐G10 and S13-‐G10 bio. During the staining we were able to bind the 3 nmol and 375 pmol

dNTP S12-‐SA-‐D7 and S12-‐SA-‐D8 to the beads. The 93,8 pmol dNTP particles weren’t able to be

differentiated from the background.

4.3 Conclusion

Of the 36 DeNAno clones with which our first project started, only sample S106-‐61 was able to

bind to the IL-‐18 beads long enough during the staining. During the titration, however, the

sensitivity of our test appeared to be approximately a 1000 times too low to detect IL -‐18 in

plasma.

The lyophilization project showed that both S12-‐SA-‐D7 and S12-‐SA-‐D8 were still able to bind to

the streptavidin beads in a staining after lyophilization. In further experiments it appeared

possible to lyophilise in epitubes with screwcaps. Furthermore, the lyophilized samples were

able to be stored at -‐20°C for the duration of one month and were still able to bind to the

streptavidin beads.

96

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3. DeNAno Technology Platform. http://www.abreos.com/#!denano-‐technology/c20m2. Published 2015.

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9. Lebel-‐Binay S, Berger A, Zinzindohoué F, et al. Interleukin-‐18: biological properties and clinical implications. Eur Cytokine Netw. 2000;11(1):15-‐26. http://www.ncbi.nlm.nih.gov/pubmed/10705295.

10. Gracie JA, Robertson SE, McInnes IB. Interleukin-‐18. J Leukoc Biol. 2003;73(2):213-‐224. doi:10.1189/jlb.0602313.

11. Ibelgaufts H. IL-‐18. http://www.copewithcytokines.de/cope.cgi?key=IL18. Published 2012.

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13. Khairnar S, Kini R, Harwalkar M, Salunkhe K, Chaudhari S. A review on freeze drying process of pharmaceuticals. Int J Res Pharm Sci. 2013;4(1):76-‐94.

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15. Timberlake KC. Chemistry, An Introduction to General, Organic, and Biological Chemistry. 12th ed. Prentice Hall; 2014.

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