Date post: | 16-Dec-2015 |
Category: |
Documents |
Upload: | brittney-moore |
View: | 217 times |
Download: | 2 times |
1SELEX
Have a random 40-mer synthesized, 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
12/1/03 11:01 PM
2
RNA
DNA
RNA
RNA
(1015)
e.g., soluble form of the affinity column material
SELEX: Systematic Evolution of Ligands by EXponential enrichment
3AMP-binding aptamer
4Streptomycin-binding aptamer
5
Tobramycin (antibiotic) bound to its aptamer (backbone)
6
Some examples of aptamer targets
Zn2ATPadenosinecyclic AMPGDP FMN (and naturally in E.coli)cocainedopamineamino acids (arginine) porphyrinbiotinorganic dyes (cibacron blue, malachite green) neutral disaccharides (cellobiose)oligopeptidesaminoglycoside antibiotics (tobramycin)proteins (thrombin, tat, rev, Factor IX, VEGF, PDGF, ricin)large glycoproteins such as CD4anthrax 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. Science 287: 820-825.
9
Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science 287: 820-825.
Aromatic ringstacking interactions
H-bonding
Specificity: Caffeine = theophilline + a methyl group on a ring N (circle); bindingis >1000 times weaker
theophilline FMN
AMP AMP
DNARNA
RNA RNA
10
Electrostatic surface map:
red= - blue = +
Base flap shuts door
11
Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science 287: 820-825.
One anti-Rev aptamer:binds peptide in alpha-helical conformation
Another anti-Rev aptamer:binds peptide in anextended conformation
MS2 protein as beta sheetbound via protruding side chains
12
DNA synthesizerT7 prom PCR site
random
1015
T7 polymerase,2’F-CTP + 2’F-UTP
2’F-RNA
Affinity chromatography selection
Enriched stableaptamer
Reverse transcriptaseNormal 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’ NH2-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
Natural enantiomers: peptides = L-amino acids nucleic acids = D-ribose
Spiegelmers for more stable RNA aptamers (spiegel = mirror)
Synthesize aD-amino acid version of your peptide target
Ordinary D-ribosenucleic acid
Synthesize the L-ribose version of thebest one
the best one
L-RNA is resistant to nucleases
the target
Noxxon (Germany)
First products:Anti-CGRPAnti-Grehlin
14Rusconi, 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 slows exonucleolytic degradation
15Kd for Factor IX = 0.6 nM
FIXa + FVIIIa cleave FX
Aptamer inhibits this activity
Conjugate to polyethylenglycol to increase bloodstream lifetime
16An antidote to stop the anti-clotting action if a patient begins to bleedJust use the complementary strand (partial) The 2 strands find each other in the bloodstream!
16-fold excess
Oligomer 5-2Ratio
An
ti-co
ag
ula
nt
act
ivity
In human plasma
17
Antidote acts fast(10 min)
Antidote lasts a long time
Tested in human serum
18
In serum of patients withheparin-induced thrombocytopenia
(can no longer use heparin)
19
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 Res 62: 4029-4033.
Aptamer vs, prostate cancer cell membrane antigen (PMSA), conjugated to rhodamine
Potential use as an anticancer diagnostic, and therapeutic.
20ORIGINAL 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 Bcovalentcross-links
Wash stringently toProduce a low background.
Stain with a protein-specificsensitive fluosecent stain(e.g, for primary amine groups)
LDH
prolactin
albumin
Somalogic, Inc.: Photoaptamers More recently:
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 sequenceGR-binding sitesecondary structure
Conserved base analysis (100’s) confirms structure
X-ray diffraction: a few 3-D structures
22
Free guanosine
23
Self-cleavage viathe hammerhead motif
Hammerhead ribozyme (self-cleavage): plant viroids and human delta virus (with Hepatitis C)
24
Hammerhead ribozyme(RNase)
Synthetic variation(cleaves in trans)
You are in charge of what it will cleave
25Model 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
Tang, J. and Breaker, R.R. 2000. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A 97: 5784-5789.
i.e., al 16 dinucleotides present as possible cleavage sites
Add Mg++RT -> cDNAPCR lots of DS-DNAT7 transcription->Lots of RNA
Proposedcleavage zone
You can use SELEX to isolate new artificial ribozymes
Proposedcleavage zone
molecules under non-permissive conditionsso they stay intact (without Mg++)
2812 different evolved ribozyme structures
Tang, J. and Breaker, R.R. 2000. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A 97: 5784-5789.
Most common = X-motif Hammerhead was one
29
Selection scheme for self-cleaving DNase DNAzymes
Solid phasestreptavidin
biotin
DNAzyme will only cleavein the presence of the cofactor(otherwise self-destructs)
Collect freed large fragment
PCR with large biotinylatedleft 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): 315-23.
DNA can also form enzymes: DNAzymes
Putative cleavage region
Pb++ and Cu++ have beendescribed
30Emilsson, G. M. and R. R. Breaker (2002). Deoxyribozymes: new activities and new applications.Cell Mol Life Sci 59(4): 596-607.
Some DNAzyme activities
over spontaneous reactionCompare 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
Soukup, G.A. and Breaker, R.R. 1999. Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96: 3584-3589.
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
Soukup, G.A. and Breaker, R.R. 1999. Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96: 3584-3589.
The same induction communication module can be used with several different allosteric aptamer modules
FMN responsive
Theo responsive ATP
responsive
34
Frauendorf, C. and Jaschke, A. 2001. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9: 2521-2524.
A theophylline-dependent ribozyme
A molecular beacon that respond to nucleic acidhybridization
Reading 2
35
Frauendorf, C. and Jaschke, A. 2001. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9: 2521-2524.
Separate substrate molecule, fluorescently tagged
quencher
36
Nearby quenching group
+
37
5X effect
Not so sensitive (0.3 mM)
H
theophylline
caffeine
38
An increasing number of DNAzyme activities are being isolated:
LigasePolymeraseDNase
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. Nature 419: 952-956.
Back to Nature: Aptamers play a role in regulation of gene expression
Thiamine:
Inhibits its own synthesis(in bacteria)
40
Shine-Delgarno sequenceribosome binding site to initiate translation
5” end ofthiM mRNA
Translation takes place
Translation initiationis inhibited
41
finis
42
Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: 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. Nature 419: 952-956.
44
Winkler, W., Nahvi, A., and Breaker, R.R. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: 952-956.