Nucleic acid aptamers

1. Nucleic acid aptamers Aptamers: 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.

2. SELEX Have a random 40-mer synthesized, between 2 arbitrary 20-mers (PCR sites) 440 = 1024 Practical limit =1015 = ~ 2 nmoles = ~ 50 ug DNA 1015is a large number.Very large (e.g., 500,000 times as many as all the unique 40-mers in the human genome.) These 1015sequences 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-mer Random 40 20-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 target of interest (protein, small molecule).

3. e.g., the soluble form of the immobilized affinity column material SELEX: Systematic Evolution of Ligands by Exponential. Enrichment for RNA (or DNA) Essential elements:1) Synthesis of randomized DNA sequences 2) In vitro T7 mediated RNA synthesis from DNA 3) Affinity chromatography 4) RT=PCR (1015) DNA RNA Ligand is immobilized here. Small molecule or large molecule. DNA RNA RNA

4. Some examples of aptamer targets Small molecules Zn2 ATP adenosine cyclic AMP GDP FMN (and an RNA aptamer is found naturally in E.coli) cocaine dopamine amino acids (arginine) porphyrin biotin organic dyes (cibacron blue, malachite green) neutral disaccharides (cellobiose, and cellulose) oligopeptides aminoglycoside antibiotics (tobramycin) Proteins thrombin HIVtat HIV rev Factor IX VEGF PDGF ricin large glycoproteins such as CD4 anthrax spores (?)

5. Electrostatic surface map:red= - blue = + Base flap shuts door

6. One anti-Rev aptamer: binds peptide in alpha-helical conformation Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science287: 820-825. Another anti-Rev aptamer: binds peptide in an extended conformation MS2 protein as beta sheet bound via protruding side chains

7. Therapeutic use of an aptamer that binds to and inhibits clotting factor IX 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. Nature419: 90-94. Reading: 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 angiplasty is heparin, which has some toxicitiy and is difficult to control. Inverted T at 3’ end (3’-3’) slows exonucleolytic degradation ( R-3’O-P-O-3’-R-T )

8. Anti-Factor IX RNA aptamer isolated by SELEX Kd for Factor IX = 0.6 nM F_IXa + F_VIIIa cleaves F_X Aptamer inhibits this activity +aptamer-PEG, Clotting time increase +aptamer+PEGylation Mutant version -aptamer == 1 Conjugate to polyethyleneglycol to increase bloodstream lifetime PEG = polyethyleneglycol polymer, appended to decrease clearance rate.

9. An 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! Antidote 5-2 design = the open squares 16-fold excess Anti-coagulant activity duplexed In human plasma free aptamer Scrambled antidote +Oligomer 5-2 Ratio of anti- to aptamer

10. Antidote acts fast (10 min) Anti-coagulant activity Need 10X antidote Antithrombin aptamer antidote tested in human serum Ratio antidote/aptamer Anti-coagulant activity Time (min) Anti-coagulant activity Antidote lasts a long time Time (hr)

11. Reduced clotting Reversed by antidote In serum of patients with heparin-induced thrombocytopenia (heparin can no longer be used)

12. Macugen: an RNA aptamer that binds VEGF and is marketed for adult macular degeneration (wet type) From the label: R Inverted ribo-T 3’-3’ to protect 3’ end 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. VEGF = vascular endothelial growth factor

13. Ribozymes = RNA enzymes 1982 Tom Cech: Tetrahymena rRNA intron is self-spliced out (Guanosine [GR] + Mg++) Altman and Pace: Ribonuclease P is an RNP: RNA component alone can process the 5’ ends of tRNAs Mitochondrial group I introns (GR –catalyzed) also can self-splice Then group II introns in mitochondria (lariat-formers) Mutations (100’s) revealed required attributes: Internal guide sequence GR-binding site secondary structure Conserved base analysis (100’s)  confirms structure X-ray diffraction: a few 3-D structures

14. lariat (natural ribozymes) Free guanosine No lariat lariat + + +

15. Point of cleavage Hammerhead ribozyme(RNase) can cleave in cis (“hammer head” is upside down) Synthetic variation:cleaves in trans You are in charge of what it will cleave(you fill in the N’s)

16. You can use SELEX to isolate new artificial ribozymes Tang, J. and Breaker, R.R. 2000. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A97: 5784-5789. 1015 DNA molecules with T7 promoter Keep molecules under non-permissive conditions so they stay intact (without Mg++) Proposedcleavage zone RT -> cDNA: Cleavage zone is rebuilt by being part of the primer. Now add Mg++ Selecting for cleavage anywhere in the zone Isolate the successfully cleaved by size on gels Proposedcleavage zone i.e., al 16 dinucleotides present as possible cleavage sites

17. New synthetic ribozymes, and DNAzymes Start with 1015 DNA molecules again Select for enzyme activity: E.g., cleaves itself off a solid support in the presence of Mg++ Many different activities have been selected.Most have to do with nucleic acid transformations;RNase, ligase, kinase, etc.But not all (C-C bond formation possible). Generally much slower than protein enzymes. Most work has been on RNases (usually associated with the word “ribozymes”)

18. Combine an aptamer and a ribozyme  Allosteric ribozyme Catalytic activity can be controlled by ligand binding ! Positive or negative. Modular Molecular switches, biosensors

19. Selection of an allosterically activated ribozyme Isolation of aptamer-ribozyme combinations that respond to ligand binding. Randomize the “communication module” Iterations Select with decreasing activation times for better and better binders. Selection of an allosterically inhibited ribozyme Soukup, G.A. and Breaker, R.R. 1999. Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A96: 3584-3589.

20. fluorophore quencher Using an allosteric ribozyme to create a chemical sensor Reading Frauendorf, C. and Jaschke, A. 2001. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem9: 2521-2524. Start with a theophylline-dependent ribozyme: Analogy: A molecular “beacon” that respond to nucleic acid hybridization

21. + Too short to maintain a stable duplex structure with SWI 58 Separate substrate molecule (in trans), fluorescently tagged Nearby quenching group kept close by hybridization

22. H theophylline 5X over background caffeine good specificity Not so sensitive (0.3 mM)

23. over spontaneous reaction Emilsson, G. M. and R. R. Breaker (2002). Deoxyribozymes: new activities and new applications.Cell Mol Life Sci59(4): 596-607. Some DNAzyme activities Compare protein enzymes, Typically 6000 on this scale (100/sec)

24. Gold, L. et al., Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery PLoS ONE, 1 December 2010, Volume 5, e15004 SomaLogic, Inc.

25. Some prominent aptamer companies: Archemix (Boston) RNA aptamers Somalogic (Colorado) DNA aptamers Noxxon (Germany) “spiegelmers”

26. siRNA = short interfering RNA Double stranded (DS) RNA miRNA = microRNA naturally occurring siRNA Dicer siRNA: ~22mer, 2 nt overhangs (Primary transcript) RISC: RNA-induced silencing complex RISC activation Single-stranded RNA Protect against viral RNA, repetitive sequence transcripts More common Anneals to mRNA target No cleavage if imperfectly complementary, but translation inhibition Cleavage if perfectly complementary mRNA target cleavage and degradation

27. miRNA = microRNA, naturally occurring siRNA Primary transcriptpoly- or mono-cistronic miRNA = microRNA naturally occurring siRNA Enzymatic processing Pre-microRNA nucleus cytoplasm Dicer Protect against viral RNA, repetitive sequence transcripts Mature miRNA Anneals to mRNA target: Cleavage if perfectly complementary Anneals to mRNA target: No cleavage if imperfectly complementary, but translation inhibition (more common)

28. 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)

29. Generation of siRNA in vitro Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer) b. 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 b. Also, can use controlled RNase to generatefragments (cheaper) Introduce perfect hairpin RNA into cells, let Dicer make siRNA Introduce imperfect hairpin RNA into cells(based on mRNA sequence) and let Dicer make miRNA

30. Limitations of exogenous siRNA treatment for silencing in mammalian cells Transient nature of the response (~3 days) Transfection problems (cell type, refractoriness) Non-renewable nature of siRNAs ($$)

31. Generation of siRNA in vivo (can give permanent knockdown) Not good for interferon- responsive cells(long DS RNA induces death response) Allow trans-association (TTTTT acts as a terminator) (Pol III) Most common, using U6 or H1 promoter U6 = small nuclear RNA used for splicing.H1 = RNA element of RNase P, used in tRNA processing. (Pol II) miRNA

32. Potential determinants of efficient siRNA-directed gene silencing 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. Delivery Transfection (lipofection, electroporation, hydrodynamic injection (mouse)) Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells)

33. Some applications: 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) 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)

34. Delivery in an intact organism Hydrodynamic injection (sudden large volume) of straight siRNA (no vector) into the tail vein of a newborn mouse Get silencing of co-injected luciferase vector in a variety of tissues High throughput siRNA for gene discovery C.elegans, 19,000 genes Make a library of 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 in an early experiment (e.g., fat metabolism phenotypes found)

35. Systemic RNAi: worms, plants, mammals In plants, get permanent post-transcriptional gene silencing (PTGS, transcriptional level) Worms: effect can last though several generations Amplified by reverse transcriptase Influx/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”

36. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054 Cotton: 20 million cotton farmers, in Asia and Africa. For every 1 kg of fiber, plant  1.65 kg seed = 21% oil, 23% protein. BUT: Seed contains the terpenoid gossypol: Which protects the plant from infections, But which is: cardiotoxic and hepatotoxic Oil is OK, but protein is contaminated with gossypol 44 million metric tons of cottonseed produced each year  9.4 million tons of protein Enough to satisfy the protein requirement of 500 million people. Terpenoid-negative cotton mutants are susceptible to infection and so are not commercially viable.

37. Delta-cadinene synthase Target the mRNA specifying the first step in gossypol synthesis

38. Recombinant plasmid T1 grows in the bacteria Agrobacterium tumefaciens which can be used as a vector for plant transfection dCS = delta-cadinene synthase shRNA neo gene terminator  alpha-globulin promoter a-globulin promoteris active only in seeds The T-DNA region of the binary vector pAGP-iHP-dCS. Arrows indicate the primers used in the PCR analyses. RB-right T-DNA border tOCS:octopine synthase terminator dCS: 604-bp d-cadinene synthase sequence pAGP: cotton a-globulin promoter (seed specific) pNOS: nopaline synthase promoter nptII:neomycin phosphotransferase II tNOS: nopaline synthase terminator LB: left T-DNA border.

39. NEO-RESISTANT TRANSFECTANT PLANTS Ten seeds from two transgenic plants from F1 of selfed matings 0.1 ug/mg PCR for transgene Note transgene-null segregants have normal gossypol levels

40. HPLC (high performance liquid chromatography) Null segregant Spots on seed indicate terpenoid glands

41. RT-PCR assay for the mRNA for the enzyme delta-cadinene synthase: Low to undetectable levels in the siRNA knocked-down plants PCR of DNA for transgene

42. Gossypol (G) and other terpenoids are NOT reduced in the leaves of transgenic plants (so resistance to infections should be normal). The same is true other aerial parts of the plant an for roots.

43. Low gossypol level analyzed through two generations of one homozygous plant were stable at 0.19 ug/mg +/- 0.013 (SEM, 50 seeds). WHO limit for human consumption is 0.6 ug/mg. ------------------------------------------------------------------------------------------------------------------------------------- Other plants could be similarly targeted: Lathyrus sativus, a hardy tropical/subtropical legume plant (neurotoxin = beta-N-oxalylamino-L-alanine) Fava beans, cassava beans: toxins = cyanogenic and contains fava glycosides (toxic to people with low levels of the enzymes glucose-6-phosphate dehydrogenease (G6PD), which is common. fava bean cassava bean