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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.

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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)

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


SELEX: Systematic Evolution of Ligands by EXponential enrichment

(1015)

RNA

DNA

RNA

RNA

e.g., soluble form of the affinity

column material





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


G-quartets dominate the structure

of antithrombin DNA aptamers


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


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


Electrostatic surface map:red= - blue = +

Base flap shuts door


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


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


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


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


Kd for Factor IX = 0.6 nM

FIXa + FVIIIa cleave FX

Aptamer inhibits this activity

Conjugate to polyethylenglycol to increase bloodstream lifetime


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


Antidote acts fast begins to bleed

(10 min)

Tested in human serum

Antidote lasts a long time


In serum of patients with begins to bleed

heparin-induced thrombocytopenia

(can no longer use heparin)


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.


ORIGINAL SELEX PAPER: conjugated to rhodamineC. 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


Ribozymes conjugated to rhodamine

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


Free guanosine conjugated to rhodamine


Hammerhead ribozyme (self-cleavage): conjugated to rhodamine

plant viroids and human delta virus (with Hepatitis C)

Self-cleavage via

the hammerhead motif


Hammerhead ribozyme conjugated to rhodamine(RNase)

Synthetic variation(cleaves in trans)

You are in charge of what it will cleave


Model of hammerhead ribozyme conjugated to rhodamine(data based)


New synthetic ribozymes, and DNAzymes conjugated to rhodamine

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”)


You can use SELEX to isolate new artificial ribozymes conjugated to rhodamine

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


12 different evolved ribozyme structures conjugated to rhodamine

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


DNA conjugated to rhodamine 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)


over spontaneous reaction conjugated to rhodamine

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)


Combine an aptamer conjugated to rhodamineand a ribozyme 

Allosteric ribozyme

Catalytic activity can be controlled by ligand binding!

Positive or negative.

Modular

Molecular switches, biosensors


Randomize the “communication module” conjugated to rhodamine

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.


The same induction communication module conjugated to rhodaminecan 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.


Reading 2 conjugated to rhodamine

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


Frauendorf, C. and Jaschke, A. 2001. conjugated to rhodamine Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem9: 2521-2524.

Separate substrate molecule, fluorescently tagged

quencher


+ conjugated to rhodamine

Nearby

quenching group


Not so sensitive conjugated to rhodamine (0.3 mM)

H

theophylline

5X effect

caffeine


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


Winkler, W., Nahvi, A., and Breaker, R.R. 2002. isolated:

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)


Translation isolated:

initiationis inhibited

Translation

takes place

5” end ofthiM mRNA

Shine-Delgarno sequenceribosome binding site to initiate translation


finis isolated:


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

Shine-Delgarno (ribosome binding site)


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


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


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