1 / 44

12/1/03 11:01 PM

12/1/03 11:01 PM. 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 is a large number. Very large (e.g., 500,000 times as many as all the unique 40-mers in the human genome.

Download Presentation

12/1/03 11:01 PM

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 12/1/03 11:01 PM 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

  2. SELEX: Systematic Evolution of Ligands by EXponential enrichment (1015) RNA DNA RNA RNA e.g., soluble form of the affinity column material

  3. AMP-binding aptamer

  4. Streptomycin-binding aptamer

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

  6. Some examples of aptamer targets Zn2 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. G-quartets dominate the structure of antithrombin DNA aptamers

  8. Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science287: 820-825.

  9. Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science287: 820-825. theophilline FMN Aromatic ringstacking interactions RNA RNA AMP AMP H-bonding RNA DNA Specificity: Caffeine = theophilline + a methyl group on a ring N (circle); bindingis >1000 times weaker

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

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

  12. 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’ NH2-YTP But not substrates for PCR enzymes. OK for T7 RNA polymerase and reverse transcriptase. So: Isolation of an RNase-resistant aptamer 1015 random DNA synthesizer PCR site T7 prom T7 polymerase, 2’F-CTP + 2’F-UTP 2’F-RNA Lots of normal DNA version Affinity chromatography selection PCR Reverse transcriptase Enriched stableaptamer Normal DNA version Normal deoxynucleoside triphosphates Final product after N iterations

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

  14. 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: Therapeutic use of an aptamer that binds to and inhibits clotting factor IX Inverted T at 3’ end slows exonucleolytic degradation

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

  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 In human plasma Anti-coagulant activity Oligomer 5-2 Ratio

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

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

  19. Aptamer vs, prostate cancer cell membrane antigen (PMSA), conjugated to rhodamine Lupold, S.E., Hicke, B.J., Lin, Y., and Coffey, D.S. 2002. Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res62: 4029-4033. Potential use as an anticancer diagnostic, and therapeutic.

  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 More recently: Somalogic, Inc.: Photoaptamers Wash stringently toProduce a low background. Stain with a protein-specificsensitive fluosecent stain(e.g, for primary amine groups) albumin prolactin LDH protein B B covalentcross-links B

  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. Free guanosine

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

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

  25. Model of hammerhead ribozyme (data based)

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

  27. 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. Proposedcleavage zone molecules under non-permissive conditionsso they stay intact (without Mg++) RT -> cDNA PCR lots of DS-DNA T7 transcription-> Lots of RNA Add Mg++ Proposedcleavage zone i.e., al 16 dinucleotides present as possible cleavage sites

  28. 12 different evolved ribozyme structures Most common = X-motif Tang, J. and Breaker, R.R. 2000. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A97: 5784-5789. Hammerhead was one

  29. DNA can also form enzymes: DNAzymes Li, Y. and R. R. Breaker (1999). "Deoxyribozymes: new players in the ancient game of biocatalysis." Curr Opin Struct Biol9(3): 315-23. Selection scheme for self-cleaving DNase DNAzymes Putative cleavage region biotin Solid phase streptavidin DNAzyme will only cleavein the presence of the cofactor(otherwise self-destructs) Pb++ and Cu++ have been described Collect freed large fragment PCR with large biotinylatedleft primer that reconstructs cleavage site(not part of the random region)

  30. 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)

  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. Randomize the “communication module” Selection of an allosterically activated ribozyme Iterations Isolation of aptamer-ribozyme combinations That respond to ligand binding. 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.

  33. The same induction communication module can be used with several different allosteric aptamer modules FMN responsive Theo responsive ATP responsive Soukup, G.A. and Breaker, R.R. 1999. Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A96: 3584-3589.

  34. Reading 2 Frauendorf, C. and Jaschke, A. 2001. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem9: 2521-2524. A theophylline-dependent ribozyme A molecular beacon that respond to nucleic acid hybridization

  35. Frauendorf, C. and Jaschke, A. 2001. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem9: 2521-2524. Separate substrate molecule, fluorescently tagged quencher

  36. + Nearby quenching group

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

  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. Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature419: 952-956. Back to Nature: Aptamers play a role in regulation of gene expression Thiamine: Inhibits its own synthesis (in bacteria)

  40. Translation initiationis inhibited Translation takes place 5” end ofthiM mRNA Shine-Delgarno sequenceribosome binding site to initiate translation

  41. finis

  42. Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature419: 952-956. Shine-Delgarno (ribosome binding site)

  43. Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature419: 952-956.

  44. Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature419: 952-956.

More Related