Recombinase Polymerase Amplification

Radhika PradhanAidan QuinnZiwei Song

Class 26_2011 updated Dec. 8, 201 12:45 AM

TwistDx Ltd.

qPCR Primer Design

Portable real-time fluorometer

ADVANTAGES AND POTENTIAL APPLICATIONS

Advantages

• From 1 molecule DNA or 10 molecules RNA to detectable levels (billions or trillions) in 5-10 min

• Low cost and simple reagents means practical applications are enormous

• Multiplexing allows simultaneous detection of multiple targets

Potential applications

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Therapeutic intervention at the level of pre-mRNA splicing

A. Interfere with improper splicing caused by splice site creation or activation

E.g., beta-thalassemia (R. Kole) in which a splice site has been created by a mutation in a hemoglobin gene

Use complementary DNA or RNA (antisense)Natural DNA/RNA rapidly degraded:

Use modified bases, sugars: PNA, morpholino, 2’ OMe,

Normally, DNA-RNA hybrids + endogenous RNase H type activity RNA destruction

Modified antisense DNA circumvents this problem (don’t want mRNA destroyed here, want to correct its splicing.)

PNA = peptide nucleic acids

Class 26_2011 updated Dec. 8, 201 12:45 AM

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B. Bias alternative splicing ratios

Target the unwanted isoform exon-intron joint.

e.g., BCL-2 isoforms, one is pro-apoptotic, one anti-apoptotic. The latter is increased in many cancers Target the anti-apoptotic isoform in cancer cells.

e.g., GABA-a-gamma-2 receptor (GABA = gamma amino butyric acid, a neurotransmitter) Long and short forms. Long form associated with mental illness.

C. Skip offensive exons

e.g., nonsense truncations in dystrophin --->

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x

Nonsense mutation truncates protein

Expendable exon (e.g., protein with many repeated domains)

Exon must be multiple of 3 in length to maintain reading frame after skipping

Antisense-induced skipping

Splicing as a target for disease therapy

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Deoxy, or also can add 2’ MOE

-O-CH2-CH2-O-CH3

Phosphorothioate deoxyoligonucleotides

MOE = methoxyethyl -

RNA modification

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Morpholino instead of deoxyribose or ribose

Modified phosphate

RNA modification for stabilization

Still base pairs OK!

ase

ase

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Attached 1 to 4 lysines here

PNA = peptide nucleic acid

Amide bonds,No ribose

B = a nucleic acid baseRNA modification

Even more extreme and more stable: peptide nucleic acids (PNAs)

Base pairs even better than natural nucleic acids (higher melting temperatures)

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Sazani P, et al. and Kole R.Systemically delivered antisense oligomers upregulate gene expression in mouse tissuesNat Biotechnol. 2002 Dec;20(12):1228-33.

EGFP: Enhanced green fluorescent protein = model system

Actin promoter, universally expressed. Induced exon skipping yields green fluorescence

Mutant globin intron has activated splice sitesAntisense “RNA” injected into tail vein, RNA was modified for stability

A. Interfere with improper splicing caused by splice site creation or activation

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Antisense treatment incell cultures (ex vivo) from themouse with the mutant EGFP gene

Control oligo (C)(50 nt downstream)was ineffective.

Max. effect = 40%

No antisense:

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Dystrophin gene 2400 kb, mRNA = 14 kb, 79 exons: a giant geneProtein maintains muscle cell membrane integrityMutation: Duchenne’s muscular dystrophySome cases (~half) are due to stop codons (nonsense) in a repetitious exon (spectrin-like repeat, length = a multiple of 3)

Deliver antisense to the ends of exon with the nonsense mutation in mdx mice (model for Duchenne’s) to promote the skipping of the nonsense-bearing exon and so avoid truncation of the protein .Use AAV (adeno-associated virus) to deliver the antisense gene

Measure:mRNA with skipped exondystrophin proteinmuscle histochemistry for dystrophin

C. Skip offensive exons

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= 3 X 71

Branch site (consensus = YNYTRAY)

protein

mRNA

79

Use antisense RNA to target the branch point upstream of the offending exon 23 and the donor splice site downstream of the exon.

BP = branch point; SD = splice donor

Sequences targeted by antisense

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U7 promoter

compl. to splice donor site compl. to branchConsensus binding site for Sm proteins (to target to pre-mRNA)

Double target synergistic (loop?) (Kole)

ITR = inverted terminal repeat, characteristic of AAV

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0 2 4 6 13 weeks

Expression of U7 antisense construct

Splicing assay (RT-PCR)

Dystrophin protein (Western)

Endog. U7 U7SmOPT-A.S.

RT-PCR

Skip exon 23, after 2-4 wks.

(slow onset =conclude slow mRNA turnover)

normal0 2 4 6 8 13 weeks

transgenic U7

included

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Top, middle ,and bottom

Normal

Untreated mdx

Treated mdx

dystrophin dystrophin-associated antigensMuscle immuno-histochemistry

intriguing

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RNAi = RNA interference

Short double stranded RNA molecules trigger thedegradation of the complementary sequence in the cell,and can inhibit translation of the targeted mRNA

Their introduction into a cell can greatly reduce any protein whose mRNA is targeted.

Inhibition is usually incomplete in mammalian cells, but can be considerable (>90%)Thus “gene knockdown” as opposed to knock-out

Alternative technologies:

Antisense RNA: block translation or splicingRibozymes: RNAs that cleave other RNAs, sequence specifically

siRNA = small inhibitory RNAsshRNA = short hairpin RNAs (both strands can be coded by one DNA)asRNA = antisense RNAmiRNA = microRNAsncRNA = noncoding RNA

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Introduction of long DS RNA into mammalian cells will trigger the “interferon response:

Cessation of protein synthesis via activation of PKR (protein kinase RNA-activated), and phosphorylation of eIF2

Global degradation of mRNA (without any sequence specificity, RNase L activation)

Spread to neighboring cells (induction and secretion of interferon)

Most small DS RNAs do not trigger this response(<30 bp)

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miRNA synthesis and maturation

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mRNA degradationInhibits translation of an mRNA

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Generation of siRNA in vitro

Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer)

Introduce perfect hairpin RNA into cells, let Dicer make siRNA

T7-mediated in vitro transcription of each complementary strand. Anneal to make long DS RNA and transfer to cells. Let Dicer make siRNA in the cell

Introduce imperfect hairpin RNA into cells(based on mRNA sequence) and let Dicer make miRNA

Also, can use controlled RNase to generatefragments (cheaper)

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TRE Tet-inducible promoter

rtTA3 Reverse tet-transactivator

UBC promoter Drives expression of rtTA3 and IRES-puro

cPPT Central Polypurine tract. Helps translocation into nucleus of non-dividing cells

WRE Enhances the stability and translation of transcripts

turbo RFP Marker to track inducible shRNAmir expression

Puror Mammalian selectable marker

AMPr Ampicillin bacterial selectable marker

5'LTR 5' long terminal repeat

pUC ori High copy replication and maintenance in e.coli

SIN-LTR 3' Self inactivating long terminal repeat

IRES Internal ribosome entry site

ZEOr Bacterial selectable marker

Generation of si RNA in vivo

Got this far

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Transient nature of the response (~3 days)

Limitations of siRNA silencing in mammalian cells

Transfection problems (cell type, refractoriness)

Can be cell type specific

Non-renewable nature of siRNAs ($$)

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siRNA

Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC.Base-pairing with mRNA.Cleavage of mRNA.

mRNA

Base-pairing with siRNA.The position of the siRNA-binding target region.Secondary and tertiary structures in mRNA.Binding of mRNA-associated proteins.The rate of mRNA translation.The number of polysomes that are associated with translating mRNA.The abundance and half-life of mRNA.The subcellular location of mRNA.

DeliveryTransfection (lipofection, electroporation, hydrodynamic injection (mouse))Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells)

Potential determinants of efficient siRNA-directed gene silencing

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Target oncogene Ras V12 (G12V) – silenced mutant ras without silencing the WT allele. Reduced the oncogenic phenotype (soft agar growth, tumor formation in nude mice)

Some applications:

T-lymphocytes infected with anti-CCR5 RNA lower levels of this HIV receptor, and lower levels of infection (5-7X)

Target an enzyme in mouse ES cells with a hairpin vector, Isolate a knockdown, make a mouse. Mouse shows same knockdown phenotype in its cells. So can target the whole mammalian organism,

Just inject a GFP silencer gene into single cell embryos of a GFP mouse:Can find a chimeric GFP mouse with reduced GFP Progeny carry it in the germ line, Get a complete knockdown mouse, without ES cells (easier)

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Hydrodynamic injection (sudden large volume) of straight siRNA (no vector) into the tail vein of a newborn mouseGet silencing of co-injected luciferase vector in a variety of tissues

C. elegans, 19,000 genes

Make a library 17,000 siRNA genes in plasmids in E. Coli.

Feed the clones of E. coli to the worms.

Look for phenotypes.

1700 genes examined for phenotypes (as of 2005)

(e.g., fat metabolism phenotypes found)

Identify the genes affected from sequnce of the siRNA

High throughput siRNA for gene discovery

Delivery in an intact organism

33NATURE 428. 2004. p. 431

tsSV40LTag inactivates p53 at 32o but not at 39o.Infect with Hu shRNA lentivirus shRNA library;select cells that grow at 39o.Knocked down genes = those necessary for p53-induced growth arrest.

Identified shRNAs

Control Control p16K.D. p53K.D. p533+p16K,K.D.

32o 39o 39o 39o 39o

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Systemic RNAi: worms, plants, mammals

In plants, get permanent post-transcriptional gene silencing (PTGS, transcriptional level)

Worms: effect can last though several generationsAmplified by reverse transcriptaseInflux/efflux via a specific transmembrane protein (in worms)

Raisons d’etre?

Infection, many viruses go through a DS RNA phase.

Repeat element silencing? (1 million Alus, + others half the human genome)Transcribed in either direction, so could form DS RNA, then RNAi inhibits action of SS ‘mRNA”

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Nature (1998) 391: 806

Discovered RNAi as they tracked down the effective agent in antisense experiments (DS RNA contaminating their SS antisense preparations had all the inhibitory activity)

Paper characterized by nice controls and variations:Several genes, whole animal phenotype, protein product (GFP), RNA level (in situs)

Phenotype of null mutant is specifically mimicked.

Introns and promoter sequences ineffective.

DS RNA from a different sequence + SS antisense RNA vs. the target: ineffective

DS RNA linked (chimeric molecule) to a single stranded portion vs, the target: ineffective

Transport of DS RNA between cells and amplification implied.

Discovery of RNA interference using double-stranded RNA

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No RNA injected

In situ hybridizations

No probe

SS antisense RNA

DS RNA

Transcript disappears (RNA degraded)

37Nucleic acid aptamersAptamers: molecules that bind other molecules with good affinity and specificity

Usually these are proteins . . . . But they can also be RNA or DNA.

That is, single stranded RNA or DNA molecules can and will fold up into secondary and tertiary structures depending on their sequence.DNA can be synthesized as very large numbers of different (random sequences)

Aptamers can be selected from among these molecules based on their ability to bind an immobilized ligand. The tiny fraction found by chance to be able to bind to your favorite ligand can by amplified by PCR (along with background molecules).

Re-iteration of the procedure will enrich for the aptamer until they dominate the population. At this point they can be cloned and sequenced.

RNA molecules can be selected by synthesizing them from a randomized DNA population using the T7 promoter appended to each DNA molecule.

This enrichment procedure is just the SELEX method described earlier for finding the RNA substrate for RNA binding proteins. In this case it’s the same procedure, looked from the opposite point of view: not what RNA will the protein bind best, but what RNA binds the protein best.

38SELEX

Have a random 40-mer synthesized, centered between 2 arbitrary 20-mers (PCR sites)

440 = 1024

Practical limit = 1015 = ~ 2 nmoles = ~ 50 ug DNA

1015 is a large number.Very large(e.g., 500,000 times as many as all the unique 40-mers in the human genome.)

These 1015 sequences are known as “sequence space”

Each DNA molecule of these 1015 (or RNA molecule copied from them) can fold into a particular 3-D structure. We know little as yet about these structures.

But we can select the molecules that bind to our target by: AFFINITY CHROMATOGRAPHY

20-merRandom 4020-mer

Previously discussed SELEX in terms of finding the substrate sequence(s) for an RNA binding protein. Here: select an RNA sequence that can bind any particular target of interest (protein, small molecule).

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Protein:thrombin

(blood protease)

RNA thrombin- binding aptamer

Who’s binding whom?

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RNA

DNA

RNA

RNA

(1015)

e.g., the soluble form of the immobilized affinity column material

SELEX: Systematic Evolution of Ligands by Exponential Enrichment . . . for RNA (or DNA)

Ligand is immobilized here.Small molecule or large molecule

Essential elements:1) Synthesis of randomized DNA sequences2) In vitro T7-mediated RNA synthesis from DNA3) Affinity chromatography4) RT-PCR

DNA

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Some examples of aptamer targets

Small molecules

Zn+2

ATPadenosinecyclic AMPGDP FMN (and an RNA aptamer is found

naturally in E.coli)cocainedopamineamino acids (arginine) porphyrinbiotinorganic dyes (cibacron blue, malachite

green) neutral disaccharides (cellobiose, and

cellulose)oligopeptidesaminoglycoside antibiotics (tobramycin)

Proteins

thrombinHIV tatHIV revFactor IX (clotting factor)VEGFPDGFricinlarge glycoproteins such as CD4anthrax spores (?)

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Electrostatic surface map:

red= - blue = +

Base flap shuts door

Tobramycin

43Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science 287: 820-825.

Another anti-Rev aptamer:binds peptide in anextended conformation

One anti-Rev aptamer:binds peptide in alpha-helical conformation

MS2 protein as beta sheet bound via protruding A.A. side chains

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Rusconi, C.P., Scardino, E., Layzer, J., Pitoc, G.A., Ortel, T.L., Monroe, D., and Sullenger, B.A. 2002.

RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419: 90-94.

Reading:

Therapeutic use of an aptamer that binds to and inhibits clotting factor IX

Inverted T at 3’ end (3’-3’) slows exonucleolytic degradation( R-3’O-P-O-3’-R-T )

Factor IX acts together with Factor VIIIa to cleave Factor X, thus activating it in a step in the blood coagulation cascade leading to a clot.

Thus inhibition of Factor IX results in inhibition of clot formation. Desirable during an angioplasty, for example.

The usual anti-coagulant used in angioplasty is heparin, which has some toxicity and is difficult to control.

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Kd for Factor IX = 0.6 nM

F_IXa + F_VIIIa cleaves F_X

4 nM aptamer inhibits this activity

Conjugate to polyethyleneglycol to increase bloodstream lifetime

mutant version

-aptamer == 1

+aptamer-PEG,

Anti-Factor IX RNA aptamer isolated by SELEX

Clotting time increase

PEG = polyethyleneglycol polymer, appended to decrease clearance rate.

+aptamer+PEGylation

46An antidote to stop the anti-clotting action if a patient begins to bleed.Would be an improvement over heparin.Just use the complementary strand (partial) as an antidote. The 2 strands find each other in the bloodstream!

16-fold excess

+Oligomer 5-2 Ratio of anti- to aptamer

An

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nt

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ivity

In human plasma duplexedfree aptamer

Antidote 5-2 design = the open squares

Scrambled antidote

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Antidote acts fast(10 min)

Antidote lasts a long time

Antithrombin aptamer antidotetested in human serum

Need 10X antidote

Ant

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Ant

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Ratio antidote/aptamer

Time (min)

Time (hr)

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In serum of patients withheparin-induced thrombocytopenia

(heparin can no longer be used)

Reduced clotting

Reversed by antidote

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R

From the label:

VEGF = vascular endothelial growth factor

Where R is and contains a PEG chain of ~ 450 ethylene glycol units.

The chemical name for pegaptanib sodium is as follows: RNA, ((2'-deoxy-2'-fluoro)C-Gm-Gm-A-A-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-Am-Gm-(2'-deoxy-2′-fluoro)U-Gm-Am-Am-(2'-deoxy-2'-fluoro)U-Gm-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)C-Gm-(3'→3')-dT), 5'-ester with α,α'-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[ω-methoxypoly(oxy-1,2-ethanediyl)], sodium salt.The molecular formula for pegaptanib sodium is C294H342F13N107Na28O188P28[C2H4O]n (where n is approximately 900) and the molecular weight is approximately 50 kilodaltons.Macugen is formulated to have an osmolality of 280-360 mOsm/Kg, and a pH of 6–7.

Inverted ribo-T3’-3’ to protect 3’ end

Macugen: an RNA aptamer that binds VEGF andis marketed for adult macular degeneration (wet type)