Transcript aptamers04

SELEX
12/1/03 11:01 PM
Have a random 40-mer synthesized, between 2 arbitrary 20-mers (PCR sites)
20-mer
Random 40
20-mer
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
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SELEX: Systematic Evolution
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of Ligands by EXponential enrichment
(1015)
RNA
DNA
RNA
RNA
e.g., soluble form of the affinity
column material
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AMP-binding aptamer
Streptomycin-binding aptamer
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Tobramycin (antibiotic) bound to its aptamer (backbone)
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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
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G-quartets dominate the structure
of antithrombin DNA aptamers
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Hermann, T. and Patel, D.J.
2000. Adaptive recognition
by nucleic acid aptamers.
Science 287: 820-825.
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Hermann, T. and Patel, D.J.
2000. Adaptive recognition
by nucleic acid aptamers.
Science 287: 820-825.
Aromatic ring
stacking interactions
theophilline
RNA
FMN
RNA
AMP
AMP
H-bonding
Specificity: Caffeine = theophilline + a
methyl group on a ring N (circle); binding
is >1000 times weaker
DNA
RNA
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Electrostatic surface map:
red= - blue = +
Base flap shuts door
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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 an
extended conformation
MS2 protein as beta sheet
bound via protruding side
chains
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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
T7 prom
PCR site
T7 polymerase,
2’F-CTP + 2’F-UTP
2’F-RNA
Lots of normal DNA version
Affinity
chromatography
selection
PCR
Reverse transcriptase
Normal DNA version
Normal deoxynucleoside
triphosphates
Enriched
stable
aptamer
Final product
after N iterations
Spiegelmers
for more stable RNA aptamers (spiegel = mirror)
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Natural enantiomers: peptides = L-amino acids
nucleic acids = D-ribose
Synthesize a
D-amino acid
version of your
peptide target
the target
Ordinary
D-ribose
nucleic acid
Synthesize the
L-ribose
version of the
best one
Noxxon (Germany)
the best one
L-RNA is resistant to nucleases
First products:
Anti-CGRP
Anti-Grehlin
Reading:
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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.
Therapeutic use of an aptamer that
binds to and inhibits clotting factor IX
Inverted T at 3’
end slows
exonucleolytic
degradation
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Kd for Factor IX = 0.6 nM
FIXa + FVIIIa cleave FX
Aptamer inhibits this activity
Conjugate to polyethylenglycol to increase
bloodstream lifetime
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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!
In human plasma
Oligomer 5-2
Anti-coagulant activity
16-fold excess
Ratio
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Tested in human serum
Antidote acts fast
(10 min)
Antidote lasts a long time
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In serum of patients with
heparin-induced thrombocytopenia
(can no longer use heparin)
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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 Res 62: 4029-4033.
Potential use as an anticancer diagnostic, and therapeutic.
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 to
Produce a low background.
albumin
Stain with a protein-specific
sensitive fluosecent stain
(e.g, for primary amine groups)
prolactin
LDH
protein
B
B
B
covalent
cross-links
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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
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Free guanosine
Hammerhead ribozyme (self-cleavage):
plant viroids and human delta virus (with Hepatitis C)
Self-cleavage via
the hammerhead motif
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Hammerhead ribozyme
(RNase)
Synthetic variation
(cleaves in trans)
You are in charge
of what it will cleave
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Model of hammerhead ribozyme
(data based)
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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”)
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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 A 97: 5784-5789.
Proposed
cleavage zone
molecules under non-permissive conditions
so they stay intact (without Mg++)
RT -> cDNA
PCR lots of DS-DNA
T7 transcription->
Lots of RNA
Add Mg++
Proposed
cleavage zone
i.e., al 16 dinucleotides
present as possible cleavage
sites
12 different evolved ribozyme structures
Most common = X-motif
Hammerhead was one
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Tang, J. and Breaker, R.R. 2000.
Structural diversity of self-cleaving ribozymes.
Proc Natl Acad Sci U S A 97: 5784-5789.
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DNA can also form enzymes: DNAzymes
Selection scheme for self-cleaving DNase DNAzymes
Putative cleavage
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.
biotin
Solid phase
streptavidin
DNAzyme will only cleave
in the presence of the cofactor
(otherwise self-destructs)
Pb++ and Cu++
have been
described
Collect freed
large fragment
PCR with large biotinylated
left primer that reconstructs
cleavage site
(not part of the random region)
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Some DNAzyme activities
Compare protein enzymes,
Typically 6000 on this scale
(100/sec)
Emilsson, G. M. and R. R. Breaker (2002).
Deoxyribozymes: new activities and
new applications.
Cell Mol Life Sci 59(4): 596-607.
over spontaneous reaction
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Combine an aptamer and a ribozyme 
Allosteric ribozyme
Catalytic activity can be controlled by ligand binding!
Positive or negative.
Modular
Molecular switches, biosensors
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Isolation of aptamer-ribozyme combinations
That respond to ligand binding.
Randomize the “communication module”
Selection of an allosterically activated ribozyme
Iterations
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 A 96: 3584-3589.
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The same induction communication module
can be used with several different
allosteric aptamer modules
FMN
responsive
Soukup, G.A. and Breaker, R.R. 1999.
Engineering precision RNA molecular switches.
Proc Natl Acad Sci U S A 96: 3584-3589.
Theo
responsive
ATP
responsive
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Reading 2
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 acid
hybridization
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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
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+
Nearby
quenching group
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H
theophylline
5X effect
caffeine
Not so sensitive
(0.3 mM)
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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
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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)
Translation
takes place
5” end of
thiM mRNA
Translation
initiation
is inhibited
Shine-Delgarno sequence
ribosome binding site to initiate translation
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finis
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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)
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Winkler, W., Nahvi, A., and Breaker, R.R. 2002.
Thiamine derivatives bind messenger RNAs
directly to regulate bacterial gene expression.
Nature 419: 952-956.
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Winkler, W., Nahvi, A., and Breaker, R.R. 2002.
Thiamine derivatives bind messenger RNAs
directly to regulate bacterial gene expression.
Nature 419: 952-956.