1Nucleic acid aptamersAptamers: molecules that bind other molecules with good affinity and specificity

Usually these are proteins . . . . But they can also be RNA or DNA.

That is, single stranded RNA or DNA molecules can and will fold up into secondary and tertiary structures depending on their sequence.DNA can be synthesized as very large numbers of different (random sequences)

Aptamers can be selected from among these molecules based on their ability to bind an immobilized ligand. The tiny fraction found by chance to be able to bind to your favorite ligand can by amplified by PCR (along with background molecules).

Re-iteration of the procedure will enrich for the aptamer until they dominate the population. At this point they can be cloned and sequenced.

RNA molecules can be selected by synthesizing them from a randomized DNA population using the T7 promoter appended to each DNA molecule.

This enrichment procedure is just the SELEX method described earlier for finding the RNA substrate for RNA binding proteins. In this case it’s the same procedure, looked from the opposite point of view: not what RNA will the protein bind best, but what RNA binds the protein best.

2SELEX

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

Previously discussed SELEX in terms of finding the substrate sequence(s) for an RNA binding protein. Here: select an RNA sequence that can bind any target of interest (protein, small molecule).

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RNA

DNA

RNA

RNA

(1015)

e.g., the soluble form of the immobilized affinity column material

SELEX: Systematic Evolution of Ligands by Exponential. Enrichment for RNA (or DNA)

Ligand is immobilized here.Small molecule or large molecule.

Essential elements:1) Synthesis of randomized DNA sequences2) In vitro T7 mediated RNA synthesis from DNA3) Affinity chromatography4) RT=PCR

DNA

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Some examples of aptamer targets

Small molecules

Zn2

ATPadenosinecyclic AMPGDP FMN (and an RNA aptamer is found

naturally in E.coli)cocainedopamineamino acids (arginine) porphyrinbiotinorganic dyes (cibacron blue, malachite

green) neutral disaccharides (cellobiose, and

cellulose)oligopeptidesaminoglycoside antibiotics (tobramycin)

Proteins

thrombinHIVtatHIV revFactor IXVEGFPDGFricinlarge glycoproteins such as CD4anthrax spores (?)

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Electrostatic surface map:red= - blue = +

Base flap shuts door

6Hermann, 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 sheet bound via protruding side chains

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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.

Reading:

Therapeutic use of an aptamer that binds to and inhibits clotting factor IX

Inverted T at 3’ end (3’-3’) slows exonucleolytic degradation( R-3’O-P-O-3’-R-T )

Factor IX acts together with Factor VIIIa to cleave Factor X, thus activating it in a step in the blood coagulation cascade leading to a clot.

Thus inhibition of Factor IX results in inhibition of clot formation. Desirable during an angioplasty, for example.

The usual anti-coagulant used in angiplasty is heparin, which has some toxicitiy and is difficult to control.

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Kd for Factor IX = 0.6 nM

F_IXa + F_VIIIa cleaves F_X

Aptamer inhibits this activity

Conjugate to polyethyleneglycol to increase bloodstream lifetime

Mutant version

-aptamer == 1

+aptamer-PEG,

Anti-Factor IX RNA aptamer isolated by SELEX

Clotting time increase

PEG = polyethyleneglycol polymer, appended to decrease clearance rate.

+aptamer+PEGylation

9An antidote to stop the anti-clotting action if a patient begins to bleed.Would be an improvement over heparin.Just use the complementary strand (partial) as an antidote. The 2 strands find each other in the bloodstream!

16-fold excess

+Oligomer 5-2 Ratio of anti- to aptamer

An

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In human plasma duplexedfree aptamer

Antidote 5-2 design = the open squares

Scrambled antidote

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Antidote acts fast(10 min)

Antidote lasts a long time

Antithrombin aptamer antidotetested in human serum

Need 10X antidote

Ant

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Ant

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Ratio antidote/aptamer

Time (min)

Time (hr)

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In serum of patients withheparin-induced thrombocytopenia

(heparin can no longer be used)

Reduced clotting

Reversed by antidote

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R

From the label:

VEGF = vascular endothelial growth factor

Where R is and contains a PEG chain of ~ 450 ethylene glycol units.

The chemical name for pegaptanib sodium is as follows: RNA, ((2'-deoxy-2'-fluoro)C-Gm-Gm-A-A-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-Am-Gm-(2'-deoxy-2′-fluoro)U-Gm-Am-Am-(2'-deoxy-2'-fluoro)U-Gm-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)C-Gm-(3'→3')-dT), 5'-ester with α,α'-[4,12-dioxo-6-[[[5-(phosphoonoxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[ω-methoxypoly(oxy-1,2-ethanediyl)], sodium salt.The molecular formula for pegaptanib sodium is C294H342F13N107Na28O188P28[C2H4O]n (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 6–7.

Inverted ribo-T3’-3’ to protect 3’ end

Macugen: an RNA aptamer that binds VEGF andis marketed for adult macular degeneration (wet type)

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Ribozymes = RNA enzymes

1982 Tom Cech: Tetrahymena rRNA intron is self-spliced out (Guanosine [GR] + Mg++)

Altman and Pace: Ribonuclease P is an 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) revealed required attributes:Internal guide sequenceGR-binding sitesecondary structure

Conserved base analysis (100’s) confirms structure

X-ray diffraction: a few 3-D structures

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Free guanosine

lariat

No lariat lariat

+ + +

(natural ribozymes)

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Hammerhead ribozyme(RNase) can cleave in cis(“hammer head” is upside down)

Synthetic variation:cleaves in trans

You are in charge of what it will cleave(you fill in the N’s)

Point of cleavage

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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.

i.e., al 16 dinucleotides present as possible cleavage sites

Now add Mg++

RT -> cDNA: Cleavage zone is rebuilt by being part of the primer.

Proposedcleavage zone

You can use SELEX to isolate new artificial ribozymes

Proposedcleavage zone

Keep molecules under non-permissive conditions so they stay intact (without Mg++)

1015 DNA molecules with T7 promoter

Selecting for cleavageanywhere in the zone

Isolate the successfully

cleaved by size on gels

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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 possible).

Generally much slower than protein enzymes.

Most work has been on RNases (usually associated with the word “ribozymes”)

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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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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

Select with decreasing activation times for better and better binders.

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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.

Start with a theophylline-dependent ribozyme:

Analogy:A molecular “beacon” that respond to nucleic acidhybridization

Reading

Using an allosteric ribozyme to create a chemical sensor

fluorophorequencher

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Nearby quenching group kept close by hybridization

+

Too short to maintain a stable duplex structure with SWI 58 Separate

substrate molecule (in trans), fluorescently tagged

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5X over background

Not so sensitive (0.3 mM)

H

theophylline

caffeine

good specificity

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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

over spontaneous reactionCompare protein enzymes,Typically 6000 on this scale (100/sec)

24Gold, L. et al., Aptamer-Based

Multiplexed Proteomic Technology for

Biomarker Discovery

PLoS ONE, 1 December 2010, Volume 5, e15004

SomaLogic, Inc.

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Some prominent aptamer companies:

Archemix (Boston) RNA aptamersSomalogic (Colorado) DNA aptamers

Noxxon (Germany) “spiegelmers”

26siRNA = short interfering RNA

siRNA:~22mer, 2 nt overhangs

RISC: RNA-induced silencing complex

More common

Single-stranded RNA

(Primary transcript)

Double stranded (DS) RNA

RISC activation

Anneals to mRNA target

mRNA target cleavage and degradation

Dicer

27miRNA = microRNA, naturally occurring siRNA

Anneals to mRNA target: Cleavage if perfectly complementary

Anneals to mRNA target: No cleavage if imperfectly complementary, but translation inhibition (more common)

Mature miRNA

Primary transcriptpoly- or mono-cistronic

Enzymatic processing

nucleus

cytoplasm

Pre-microRNA

Dicer

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Introduction of long DS RNA into mammalian cells will trigger the “interferon response”:

Cessation of protein synthesis via activation of PKR (protein kinase, RNA-activated) and phosphorylation of eIF2

Global degradation of mRNA without any sequence specificity (RNase L activation)

Spread to neighboring cells (induction and secretion of interferon)

Most small DS RNAs do not trigger this response(<30 bp)

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Generation of siRNA in vitro

Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer)

Introduce perfect hairpin RNA into cells,let Dicer make siRNA

b. T7-mediated in vitro transcription of each complementary strand. Anneal to make long DS RNA and transfer to cells. Let Dicer make siRNA in the cell

Introduce imperfect hairpin RNA into cells(based on mRNA sequence) and let Dicer make miRNA

b. Also, can use controlled RNase to generatefragments (cheaper)

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Transient nature of the response (~3 days)

Limitations of exogenous siRNA treatment for silencing in mammalian cells

Transfection problems (cell type, refractoriness)

Non-renewable nature of siRNAs ($$)

31Generation of siRNA in vivo (can give permanent knockdown)

Allow trans-association(TTTTT acts as a terminator)

Not good for interferon-responsive cells(long DS RNA induces death response)

miRNA

Most common, using U6 or H1 promoterU6 = small nuclear RNA used for splicing.H1 = RNA element of RNase P, used in tRNA processing.

(Pol III)

(Pol II)

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siRNA

Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC.Base-pairing with mRNA.Cleavage of mRNA.

mRNA

Base-pairing with siRNA.The position of the siRNA-binding target region.Secondary and tertiary structures in mRNA.Binding of mRNA-associated proteins.The rate of mRNA translation.The number of polysomes that are associated with translating mRNA.The abundance and half-life of mRNA.The subcellular location of mRNA.

DeliveryTransfection (lipofection, electroporation, hydrodynamic injection (mouse))Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells)

Potential determinants of efficient siRNA-directed gene silencing

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Target oncogene Ras V12 (G12V) – silenced mutant ras without silencing the WT allele. Reduced the oncogenic phenotype (soft agar growth, tumor formation in nude mice)

Some applications:

T-lymphocytes infected with anti-CCR5 RNA lower levels of this HIV receptor, and lower levels of infection (5-7X)

Target an enzyme in mouse ES cells with a hairpin vector, Isolate a knockdown, make a mouse. Mouse shows same knockdown phenotype in its cells. So can target the whole mammalian organism,

Just inject a GFP silencer gene into single cell embryos of a GFP mouse:Can find a chimeric GFP mouse with reduced GFP Progeny carry it in the germ line, Get a complete knockdown mouse, without ES cells (easier)

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Hydrodynamic injection (sudden large volume) of straight siRNA (no vector) into the tail vein of a newborn mouseGet silencing of co-injected luciferase vector in a variety of tissues

C.elegans, 19,000 genes

Make a library of 17,000 siRNA genes in plasmids in E. Coli.

Feed the clones of E. coli to the worms.

Look for phenotypes.

1700 genes examined for phenotypes in an early experiment

(e.g., fat metabolism phenotypes found)

High throughput siRNA for gene discovery

Delivery in an intact organism

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Systemic RNAi: worms, plants, mammals

In plants, get permanent post-transcriptional gene silencing (PTGS, transcriptional level)

Worms: effect can last though several generationsAmplified by reverse transcriptaseInflux/efflux via a specific transmembrane protein (in worms)

Raisons d’etre?

Infection, many viruses go through a DS RNA phase.

Repeat element silencing? (1 million Alus, + others half the human genome)Transcribed in either direction, so could form DS RNA, then RNAi inhibits action of SS ‘mRNA”

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Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054

Cotton: 20 million cotton farmers, in Asia and Africa.For every 1 kg of fiber, plant 1.65 kg seed = 21% oil, 23% protein.BUT:Seed contains the terpenoid gossypol:Which protects the plant from infections,

But which is:cardiotoxic and hepatotoxic

Oil is OK, but protein is contaminated with gossypol

44 million metric tons of cottonseed produced each year 9.4 million tons of proteinEnough to satisfy the protein requirement of 500 million people.

Terpenoid-negative cotton mutants are susceptible to infection and so are not commercially viable.

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Target the mRNA specifying the first step in gossypol synthesis

Delta-cadinene synthase

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dCS = delta-cadinene synthase

The T-DNA region of the binary vector pAGP-iHP-dCS. Arrows indicate the primers used in the PCR analyses.RB-right T-DNA bordertOCS:octopine synthase terminatordCS: 604-bp d-cadinene synthase sequencepAGP: cotton a-globulin promoter (seed specific)pNOS: nopaline synthase promoternptII:neomycin phosphotransferase IItNOS: nopaline synthase terminatorLB: left T-DNA border.

alpha-globulin promoter

terminator

neo gene

a-globulin promoteris active only in seeds

Recombinant plasmid T1 grows in the bacteria Agrobacterium tumefacienswhich can be used as a vector for plant transfection

shRNA

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Ten seeds from two transgenic plants from F1 of selfed matings

PCR for transgene

Note transgene-null segregants have normal gossypol levels

0.1 ug/mg

NEO-RESISTANT TRANSFECTANT PLANTS

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Spots on seed indicate terpenoid glands

Null segregant

HPLC (high performance liquid chromatography)

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RT-PCR assay for the mRNA for the enzyme delta-cadinene synthase:Low to undetectable levels in the siRNA knocked-down plants

PCR of DNA for transgene

42

Gossypol (G) and other terpenoids are NOT reduced in the leaves of transgenic plants(so resistance to infections should be normal). The same is true other aerial parts of the plant an for roots.

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Low gossypol level analyzed through two generations of one homozygous plant were stable at 0.19 ug/mg +/- 0.013 (SEM, 50 seeds).

WHO limit for human consumption is 0.6 ug/mg.-------------------------------------------------------------------------------------------------------------------------------------Other plants could be similarly targeted:

Lathyrus sativus, a hardy tropical/subtropical legume plant (neurotoxin = beta-N-oxalylamino-L-alanine)

Fava beans, cassava beans: toxins = cyanogenic and contains fava glycosides (toxic to people with low levels of the enzymes glucose-6-phosphate dehydrogenease (G6PD), which is common.

fava beancassava bean