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New Product:X-Aptamer Selection Kit
Updated April 2015
Technology Basics
X-Aptamers are synthetic affinity molecules Similar in function to monoclonal antibodies
X-Aptamers combine a variety of chemical moieties DNA/RNA, amino acid groups, and small molecules
Chemical diversity is far superior to standard aptamers
Improves target interaction for better binding affinity and specificity
A unique bead-based process is used to select X-Aptamers Only basic molecular biology techniques and equipment required
Enables the X-Aptamer Selection Kit
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Affinity Molecule Comparison
3
Key Attributes Antibodies Regular Aptamers X-Aptamers
Affinity Excellent Very Good Excellent
Specificity Excellent Very Good Excellent
Size & Complexity High Low Low
Discovery In Vivo In Vitro In Vitro
Storage Refrigerated, Frozen Ambient Ambient
Shelf Life Months Years Years
Production Biological Chemical Chemical
Batch Reproducibility Low High High
Functionalizing Complicated Simple and Vast Simple and Vast
Discovery Slow Slow Rapid
X-Aptamer Chemical Diversity: Examples
Base
X-Aptamers Can Use a Virtually Unlimited Array of Chemical Functionalities
• Incorporated prior to the selection process• Combinations of multiple functionalities easily accomplished• Many are incompatible with SELEX (the solution-based aptamer selection process)
R
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Phosphorodithioate
TargetRegular Aptamer
KD, unmodified
Modification Made
X-AptamerKD, modified
Binding Affinity
Enhancement
VEGF165 2.1 nMPS2
FS2
15 pM
2.3 pM
140x
913x
α-Thrombin 1.1 nM PS2 1.2 pM 916x
α-Thrombin 2.4 nM T-indole 1.4 pM 1700x
IgE 11.3 nM PS2 10.4 pM 1000x
Increased Chemical Diversity = Better Performance
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New Product: X-Aptamer Selection Kit
Enables customer to easily select X-Aptamers
Selection conditions match application
Target identity can remain confidential
Up to 3 targets simultaneously
No special equipment required
Rapid, straightforward, and effective
This is NOT the cumbersome SELEX process!
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Purchase Kit
Perform Selection
(up to 3 targets)
Send Sample to
AM Biotech
Identify X-Aptamer
Sequences
Synthesize
X-Aptamers
Send X-Aptamers to
Customer
Customer Uses
X-Aptamers
<1 w
eek
~3 w
eeks
Cu
sto
me
rA
M B
iote
ch
~4 W
eeks
X-Aptamer Selection Kit: Complete Flow
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Bead Library Production
Perform Selection Protocol with up to 3
Desired Targets
Data Analysis
AM BIOTECHNOLOGIES CUSTOMER
Next Generation Sequencing
Kit
Sample
X-AptamersKit purchase includes a
license for unlimited use of X-Aptamers for research.
Synthesize X-Aptamers
Test & Use X-Aptamer(s)
material only
Licensing terms are available for using X-Aptamers in a commercial product or service.
Unique Reagent Production
Kit Testing Results
Prototype X-Aptamer Selection Kits tested USA, Canada, India, South Africa
100% successful Every completed kit produced X-Aptamers to at least one target
Each kit could process up to three targets simultaneously
~70% of all targets attempted were successful
Success rate exceeds the SELEX process SELEX overall success rate estimated to be 30% to 50%
SELEX process is used to select conventional aptamers
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What Prototype Kit Users Said
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“Using the prototype kit we were able to select nanomolar affinity X-Aptamers to a target candidate biomarker of Tuberculosis disease…that we failed to raise DNA aptamers to via conventional SELEX procedures and we believe that the chemical modification of the X-Aptamer library was key to this success.” – Dr. Jonathan B., University of Cape Town
“The ease of use [of the kit] allowed my undergraduate researchers to be able to skillfully perform the selection experiments, and we have since identified one of the putative aptamers identified as a success – this aptamer binds with low nanomolar affinity and is being used to generate an electrochemical biosensor platform for the detection of botulism.” – Dr. Andrew B., Metropolitan State University of Denver
“Compared to aptamers selected through SELEX protocol, X-Aptamers generated through this kit demonstrated much higher affinity to their target molecules on our platform.” – Dr. Renuka S., University of North Carolina at Greensboro
“Working in an aptamer research lab, I’m regularly approached by companies and researchers from all around the world for technical help with their aptamer selections. It seems to me that there has been a demand for aptamers-on-demand for quite some time and it is delightful to see it is finally becoming a reality!” – Dr. Gwendolyn S., University of Texas at Austin
Kit Pricing – NOT FOR PUBLIC DISCLOSURE
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Kit Processing Steps Price* Comments
X-Aptamer Selection Kit Purchase $500 Enables multiplex selection using up to 3 targets
Quality control markers indicate if kit was successfully processed
Next Gen Sequencing (NGS) and Data Analysis
NGS and data analysis by AM Biotech
-or-
Data analysis only by AM Biotech
$2,500
-or-
$1,250
Customers may choose to obtain NGS from their own vendor/core laboratory
Ion Torrent PGM 316/318 chip is preferred platform (FASTQ file required)
Data analysis ranks the sequences for the likelihood of binding to each target
Data analysis may also generally indicate sequence specificity to its target
Putative X-Aptamer Synthesis
First 15 (total to all targets attempted)
Second 15 - Optional
Additional (per sequence) – Optional
$2,250
$1,875
$ 100
Synthesize and desalt sequences that data analysis identifies as likely X-Aptamers
Putative X-Aptamers can be unlabelled or labeled with either biotin or amine
Other labels available for an additional charge
X-Aptamer affinity at this stage is expected to be in the nanomolar range
X-Aptamer Enhancement Service – Optional
(performed by AM Biotech) Request Quote
Sequence truncation and PS2 backbone modifications to enhance binding affinity
100X to >1000X affinity enhancement is realistic
Affinity of resulting PS2 X-Aptamers expected to be in the picomolar range
Commercial use license Varies by
Market
Customer receives a license for unlimited research use of X-Aptamers developed
A commercial use license from AM Biotech is required prior to using any X-Aptamer
in a commercial product/service or clinical trial
Large-scale X-Aptamer Synthesis Request Quote
AM Biotech can synthesize X-Aptamers at the multi-gram or larger scale
Third party synthesis can be performed after obtaining a commercial use license
* Prices subject to change.
Total Price of Kit-based X-Aptamer Selection
$500 Purchase Kit
$2,500-$1,250
AM Biotech Next Generation Sequencing & Data AnalysisAM Biotech Data Analysis Only (customer arranges NGS)
$2,250Synthesis of 15 Putative X-Aptamers (five per target assuming three targets)
$4,000 to $5,250 Total Customer Outlay
$1,333 to $1,750 TOTAL PRICE PER TARGET*(assuming 3 targets attempted)
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* Compared to competitors’ prices of $10,000 to $25,000 per target for turnkey selection of a conventional aptamer using SELEX .
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Contacts
Mark ShumberaPresident(832) [email protected]
Tim McGrathDirector, Business Development(713) [email protected]
AM Biotechnologies, LLC12521 Gulf FreewayHouston, Texas 77034-4509
www.am-biotech.com
Additional Information
The following slides provide a brief overview of the technology underlying the X-Aptamer Selection Kit.
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Basic Components of X-Aptamer Selection
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Two-step Bead & Solution Screening Against Targets Sequencing
Choose & Synthesize
X-Aptamers
Synthesize Library with
Unique Reagents
Selection Process
Progression
X-APTAMER LIBRARY
Library: Key Enabler of X-Aptamers and the Kit
A library of billions of different DNA sequences is synthesized on microbeads
Total library mass/volume: ~100 mg/0.5 cc
Sequence diversity: 108 to 1010
Each microbead has ~0.5 picomoles of a unique DNA sequence attached
15
300 µm
Library Microbead Example
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5’ Primer 5’ Stem Combinatorial (20-40N)
-NNNNNNNNNNNNNNNNNNNNNNNNN-
N = natural or chemically modified nucleotide;nature of modification is position-dependent
Notional Combinatorial Region on One Bead*-UTCGAAAUCTGGGACCGUGTTCGUA-
Black = standard DNAGreen= phosphorodithioate
Red = 2’-OMe phosphorodithioateBlue = X-modified dU (one or more)
Examples of AvailabledU X-ModificationsIndole (Tryptophan)
Phenol (Tyrosine)Guanidine (Arginine)
SerineBoron
Small moleculesOthers
3’ Stem 3’ Primer
used to help sequence the DNA strand
* Technical Note: The oligonucleotide that includes the chemical modifications shown above cannot be amplified using PCR; however, an unmodified DNA sequence can be recovered. If this bead is selected from the library, AM Biotech uses the unmodified DNA sequence recovered after NGS as a ‘barcode’ to re-synthesize the oligonucleotide attached to this bead with the appropriate chemical modifications in the correct sequence positions.
Combinatorial Library Synthesis
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A pool and split process (left) creates many copiesof a unique DNA sequence on each bead.
A DNA base is added to each of four synthesiscolumns. All the beads in one column get the sameDNA base added. The beads are then removedfrom all of the columns, mixed together, andrandomly redistributed back into the four columns.Another base is added. This process is repeatednumerous times.
This combinatorial process is performed on apatented instrument and can create as manyunique DNA sequences in parallel as there arebeads available.
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incubate library + tagged protein
G
A
GT
T
A
AC
G
T
X
A
C
G
TX
A
C
G
T
X
A
C
GT
X
G
ATC
GC
G
A
GT
T
A
AC
G
T
X
A
C
G
TX
A
C
G
T
X
A
C
GT
X
G
ATC
GC
protein binds beadswith high affinity
sequences
anti-tag magneticparticles bind
protein
first stagemagnetic selection
of library beads
recoveredbeads (true
& false positives)
cleave sequences frombeads into solution
second-stagesolution pull-downwith target protein
next gen sequencing of solution pull-
down
resynthesize high-affinity X-Aptamers
Bead-based Selection Process
Combinatorial bead library
(one bead/one sequence)
analyze sequences to identify X-Aptamers
Selection process steps shaded in blue are performed by the customer using the kit.
Following slides provide additional detail.
Kit Protocol 1st Stage
Add library beads for selection
Magnetic selection
of library beads
Isolate magnetically recovered
beads
Incubate up to 3 tagged targets with anti-tag magnetic
particles
Cleave sequences from
beads into solution
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GO TO NEXT SLIDE
START KIT PROTOCOL
Kit Protocol 2nd Stage
Split into 5 fractions
Start pool control
Add Target 1
Add Target 2
Add Target 3
No target controlA
dd
an
ti-t
ag m
agn
etic
par
ticl
es
Iso
late
bin
der
s b
y m
agn
etic
pu
ll-d
ow
n
Recombine for next
generation sequencing
PC
R a
mp
lify
wit
h b
ar-c
od
ed p
rim
ers
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END OF KIT PROTOCOL
CONTINUATION FROM PREVIOUS
SLIDE
