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

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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

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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:

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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 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

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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)

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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

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.