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12/1/03 1101 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 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-mer
Random 40
20-mer
corrected, switching with Merrill Velez, 12/11/05
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SELEX Systematic Evolution of Ligands by
EXponential enrichment
(1015)
RNA
DNA
RNA
RNA
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Typical RNA aptamer
AMP-binding aptamer
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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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An example of tertiary structure
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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theophilline
FMN
AMP
Hermann, T. and Patel, D.J.2000. Adaptive
recognition by nucleic acid aptamers. Science
287 820-825.
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 gt1000
times weaker
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Electrostatic surface mapred - 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.
MS2 protein as beta sheet bound via protruding
side chains
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Stabilizing RNA aptamers 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. But yes 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, 2F-CTP 2F-UTP
2F-RNA
Lots of normal DNA version
Affinity chromatography selection
PCR
Enriched stableaptamer
Reverse transcriptase
Normal DNA version
Normal deoxynucleoside triphosphates
Final product after N iterations
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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.
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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. Nature
419 90-94.
Reading
Inverted T at 3 end (3-3) slows exonucleolytic
degradation ( R-3O-P-O-3-R-T )
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Kd for Factor IX 0.6 nM
FIXa FVIIIa cleave FX
Aptamer inhibits this activity
aptamer
Mutant version
-aptamer
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!
Antidote 5-2 open squares
16-fold excess
duplexed
In human plasma
free aptamer
Anti-coagulant activity
Scrambled antidote
Oligomer 5-2
Ratio of anti- to aptamer
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Tested in human serum
Need 10X antidote
Antidote lasts a long time
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Reduced clotting
Reversed by antidote
In serum of patients with heparin-induced
thrombocytopenia (can no longer use heparin)
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Macugen an RNA aptamer that bonds VEGF and Is
marketed for adult macular degeneration (wet type)
From the label
R
Where R is and n is
approximately 450. The chemical name for
pegaptanib sodium is as follows RNA,
((2'-deoxy-2'-fluoro)C-Gm-Gm-A-A-(2'-deoxy-2'-flu
oro)U-(2'-deoxy-2'-fluoro)C-Am-Gm-(2'-deoxy-2'-flu
oro)U-Gm-Am-Am-(2'-deoxy-2'-fluoro)U-Gm-(2'-deoxy-
2'-fluoro)C-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-flu
oro)U-Am-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-flu
oro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro
)C-(2'-deoxy-2'-fluoro)C-Gm-(3'?3')-dT), 5'-ester
with a,a'-4,12-dioxo-6-5-(phosphoonoxy)pentyl
aminocarbonyl-3,13-dioxa-5,11-diaza-1,15-pentade
canediylbis?- methoxypoly(oxy-1,2-ethanediyl),
sodium salt. The molecular formula for
pegaptanib sodium is C294H342F13N107Na28O188P28C2
H4On (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 67.
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DNA aptamers for protein diagnostics
Photoaptamers, from Somalogic, Inc.
ORIGINAL SELEX PAPERC. Tuerk and L. Gold.
"Systematic evolution of ligands by exponential
enrichment RNA ligands to bacteriophage T4 DNA
polymerase," Science, 249505-10, 1990
Make DNA aptamers using bromouracil instead of
thymine Bromouracil absorbs near UV light (313
nm) (Normal DNA absorbs at 260 nm) Apply blood
to a photo-aptamer macroarray
Wash stringently toproduce a low
background (e.g., NaOH). Stain with a
fluorescent stainagainst proteins (e.g, for
primary amine groups)
albumin
prolactin
LDH
protein
B bromouracil bases
B
B
covalentcross-links
B
Different aptamers (many molecules per spot)
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Ribozymes 1982 Cech
Tetrahymena rRNA intron is self-spliced out
(Guanosine 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 (100s)
revealed Internal guide sequence GR-binding
site secondary structure Conserved base
analysis (100s) ? confirms structure X-ray
diffraction a few 3-D structures
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Free guanosine
No lariat
lariat
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Natural ribozyme 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) (hammer head is
upside down)
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 transformationsRNase, 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.
1015 DNA molecules with T7 promoter
Keep molecules under non-permissive conditions
so they stay intact (without Mg)
Proposedcleavage zone
RT -gt cDNA PCR lots of DS-DNA T7
transcription-gt Lots of RNA
Now add Mg
Selecting for cleavage anywhere in the zone
Proposedcleavage zone
i.e., al 16 dinucleotides present as possible
cleavage sites
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12 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.
Hammerhead was one
Common substrate (all 16 dinucleotide combos)
Rate (min-1)
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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 Biol
9(3) 315-23.
Selection scheme for self-cleaving DNase DNAzymes
Putative cleavage region
biotin
Solid phase streptavidin
Selecting for DNAzymes that will only cleave in
the presence of the cofactor (otherwise Will
self-destruct)
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)
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Emilsson, G. M. and R. R. Breaker (2002).
Deoxyribozymes new activities and new
applications.Cell Mol Life Sci 59(4) 596-607.
Some DNAzyme activities
Compare protein enzymes, Typically 6000 on this
scale (100/sec)
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Combine an aptamer and a ribozyme ? Allosteric
ribozyme Catalytic activity can be controlled by
ligand binding! Positive or negative. Modular M
olecular switches, biosensors
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Selection scheme for an allosteric (regulatable)
self-cleaving DNase DNAzyme
Ligand binding domain
Communication domain
Catakytic domain
DNAzyme will only cleavein the presence of the
cofactor (for activator) or in the absence of
the Cofactor (for inhibitor)
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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 A 96 3584-3589.
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The same induction communication module can be
used with several different allosteric aptamer
modules
FMN responsive
Theo responsive
ATP responsive
With ligand Without ligand
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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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 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
(only active in the presence of the ligand
theophylline)
quencher
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Too short to maintain a stable duplex structure
Nearby quenching group
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H
theophylline
5X effect
caffeine
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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)
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Translation takes place
Shine-Delgarno sequenceribosome binding site to
initiate translation
5 end ofthiM mRNA
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Multiplexed Amplified Aptamer-Based Real-Time
Biosensor (deals with the issue of poor catalytic
rate for aptazymes)
Fluorogenic trypsin substrate (AMC-peptide)
Cu
trypsin

Copper-responsive aptazyme(Carmi and Breaker)
Next allosteric aptazyme responsive to TNT
Shifang Zhang, P. Somasondaran and L.A. Chasin,
2005
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finis
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(No Transcript)
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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.
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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-G
rehlin
the best one
L-RNA is resistant to nucleases