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1 SELEX Have a random 40-mer synthesized, between 2 arbitrary 20-mers (PCR sites) 4 40 = 10 24 Practical limit = 10 15 = ~ 2 nmoles = ~ 50 ug DNA 10 15.

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Presentation on theme: "1 SELEX Have a random 40-mer synthesized, between 2 arbitrary 20-mers (PCR sites) 4 40 = 10 24 Practical limit = 10 15 = ~ 2 nmoles = ~ 50 ug DNA 10 15."— Presentation transcript:

1 1 SELEX Have a random 40-mer synthesized, between 2 arbitrary 20-mers (PCR sites) 4 40 = Practical limit = = ~ 2 nmoles = ~ 50 ug DNA is a large number. Very large (e.g., 500,000 times as many as all the unique 40-mers in the human genome. These sequences are known as “sequence space” Each DNA molecule of these (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 12/1/03 11:01 PM

2 2 RNA DNA RNA (10 15 ) e.g., soluble form of the affinity column material SELEX: Systematic Evolution of Ligands by EXponential enrichment

3 3 AMP-binding aptamer

4 4 Streptomycin-binding aptamer

5 5 Tobramycin (antibiotic) bound to its aptamer (backbone)

6 6 Some examples of aptamer targets Zn 2 ATP adenosine cyclic AMP GDP FMN (and naturally in E.coli) cocaine dopamine amino acids (arginine) porphyrin biotin organic dyes (cibacron blue, malachite green) neutral disaccharides (cellobiose) oligopeptides aminoglycoside antibiotics (tobramycin) proteins (thrombin, tat, rev, Factor IX, VEGF, PDGF, ricin) large glycoproteins such as CD4 anthrax spores

7 7 G-quartets dominate the structure of antithrombin DNA aptamers

8 8 Hermann, T. and Patel, D.J Adaptive recognition by nucleic acid aptamers. Science 287:

9 9 Aromatic ring stacking interactions H-bonding Specificity: Caffeine = theophilline + a methyl group on a ring N (circle); binding is >1000 times weaker theophilline FMN AMP DNA RNA

10 10 Electrostatic surface map: red= - blue = + Base flap shuts door

11 11 Hermann, T. and Patel, D.J Adaptive recognition by nucleic acid aptamers. Science 287: One anti-Rev aptamer: binds peptide in alpha-helical conformation Another anti-Rev aptamer: binds peptide in an extended conformation MS2 protein as beta sheet bound via protruding side chains

12 12 DNA synthesizer T7 prom PCR site random T7 polymerase, 2’F-CTP + 2’F-UTP 2’F-RNA Affinity chromatography selection Enriched stable aptamer Reverse transcriptase Normal DNA version PCR Lots of normal DNA version Final product after N iterations RNA aptamers are unstable in vivo (bloodstream) DNA aptamers are more stable but still can be destroyed by DNases. Modification to protect: 2’ F-YTP (Y = pyrimidine) 2’ NH 2 -YTP But not substrates for PCR enzymes. OK for T7 RNA polymerase and reverse transcriptase. So: Isolation of an RNase-resistant aptamer Normal deoxynucleoside triphosphates

13 13 Natural enantiomers: peptides = L-amino acids nucleic acids = D-ribose Spiegelmers for more stable RNA aptamers (spiegel = mirror) Synthesize a D-amino acid version of your peptide target Ordinary D-ribose nucleic acid Synthesize the L-ribose version of the best one the best one L-RNA is resistant to nucleases the target Noxxon (Germany) First products: Anti-CGRP Anti-Grehlin

14 14 Rusconi, C.P., Scardino, E., Layzer, J., Pitoc, G.A., Ortel, T.L., Monroe, D., and Sullenger, B.A RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419: Reading: Therapeutic use of an aptamer that binds to and inhibits clotting factor IX Inverted T at 3’ end slows exonucleolytic degradation

15 15 Kd for Factor IX = 0.6 nM FIXa + FVIIIa cleave FX Aptamer inhibits this activity Conjugate to polyethylenglycol to increase bloodstream lifetime

16 16 An antidote to stop the anti-clotting action if a patient begins to bleed Just use the complementary strand (partial) The 2 strands find each other in the bloodstream! 16-fold excess Oligomer 5-2 Ratio Anti-coagulant activity In human plasma

17 17 Antidote acts fast (10 min) Antidote lasts a long time Tested in human serum

18 18 In serum of patients with heparin-induced thrombocytopenia (can no longer use heparin)

19 19 Lupold, S.E., Hicke, B.J., Lin, Y., and Coffey, D.S Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res 62: Aptamer vs, prostate cancer cell membrane antigen (PMSA), conjugated to rhodamine Potential use as an anticancer diagnostic, and therapeutic.

20 20 ORIGINAL SELEX PAPER: C. Tuerk and L. Gold. "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase," Science, 249:505-10, 1990 protein B B B covalent cross-links Wash stringently to Produce a low background. Stain with a protein-specific sensitive fluosecent stain (e.g, for primary amine groups) LDH prolactin albumin Somalogic, Inc.: Photoaptamers More recently:

21 21 Ribozymes 1982 Cech: Tetrahymena rRNA intron is self-spliced out (GR + Mg++) Altman and Pace: Ribonuclease P 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): Internal guide sequence GR-binding site secondary structure Conserved base analysis (100’s)  confirms structure X-ray diffraction: a few 3-D structures

22 22 Free guanosine

23 23 Self-cleavage via the hammerhead motif Hammerhead ribozyme (self-cleavage): plant viroids and human delta virus (with Hepatitis C)

24 24 Hammerhead ribozyme (RNase) Synthetic variation (cleaves in trans) You are in charge of what it will cleave

25 25 Model of hammerhead ribozyme (data based)

26 26 New synthetic ribozymes, and DNAzymes Start with 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). Generally much slower than protein enzymes. Most work has been on RNases (usually associated with the word “ribozymes”)

27 27 Tang, J. and Breaker, R.R Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A 97: i.e., al 16 dinucleotides present as possible cleavage sites Add Mg++ RT -> cDNA PCR lots of DS-DNA T7 transcription-> Lots of RNA Proposed cleavage zone You can use SELEX to isolate new artificial ribozymes Proposed cleavage zone molecules under non-permissive conditions so they stay intact (without Mg++)

28 28 12 different evolved ribozyme structures Tang, J. and Breaker, R.R Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A 97: Most common = X-motif Hammerhead was one

29 29 Selection scheme for self-cleaving DNase DNAzymes Solid phase streptavidin biotin DNAzyme will only cleave in the presence of the cofactor (otherwise self-destructs) Collect freed large fragment PCR with large biotinylated left primer that reconstructs cleavage site (not part of the random region) Li, Y. and R. R. Breaker (1999). "Deoxyribozymes: new players in the ancient game of biocatalysis." Curr Opin Struct Biol 9(3): DNA can also form enzymes: DNAzymes Putative cleavage region Pb++ and Cu++ have been described

30 30 Emilsson, G. M. and R. R. Breaker (2002). Deoxyribozymes: new activities and new applications. Cell Mol Life Sci 59(4): Some DNAzyme activities over spontaneous reaction Compare protein enzymes, Typically 6000 on this scale (100/sec)

31 31 Combine an aptamer and a ribozyme  Allosteric ribozyme Catalytic activity can be controlled by ligand binding! Positive or negative. Modular Molecular switches, biosensors

32 32 Soukup, G.A. and Breaker, R.R Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96: Selection of an allosterically inhibited ribozyme Isolation of aptamer-ribozyme combinations That respond to ligand binding. Randomize the “communication module” Selection of an allosterically activated ribozyme Iterations

33 33 Soukup, G.A. and Breaker, R.R Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96: The same induction communication module can be used with several different allosteric aptamer modules FMN responsive Theo responsive ATP responsive

34 34 Frauendorf, C. and Jaschke, A Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9: A theophylline-dependent ribozyme A molecular beacon that respond to nucleic acid hybridization Reading 2

35 35 Frauendorf, C. and Jaschke, A Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9: Separate substrate molecule, fluorescently tagged quencher

36 36 Nearby quenching group +

37 37 5X effect Not so sensitive (0.3 mM) H theophylline caffeine

38 38 An increasing number of DNAzyme activities are being isolated: Ligase Polymerase DNase And activities using co-enzymes, as protein enzymes do: E.g., co-enzyme A

39 39 Winkler, W., Nahvi, A., and Breaker, R.R Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: Back to Nature: Aptamers play a role in regulation of gene expression Thiamine: Inhibits its own synthesis (in bacteria)

40 40 Shine-Delgarno sequence ribosome binding site to initiate translation 5” end of thiM mRNA Translation takes place Translation initiation is inhibited

41 41 finis

42 42 Winkler, W., Nahvi, A., and Breaker, R.R Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: Shine-Delgarno (ribosome binding site)

43 43 Winkler, W., Nahvi, A., and Breaker, R.R Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419:

44 44 Winkler, W., Nahvi, A., and Breaker, R.R Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419:


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