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Nucleic acid aptamers Aptamers: molecules that bind other molecules with good affinity and specificity Usually these are proteins . . . . But they can also be RNA or DNA.

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Nucleic acid aptamers

Aptamers: 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.


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

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).


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

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

Essential elements:1) Synthesis of randomized DNA sequences

2) In vitro T7 mediated RNA synthesis from DNA

3) Affinity chromatography

4) RT=PCR

(1015)

DNA

RNA

Ligand is immobilized here.

Small molecule or large molecule.

DNA

RNA

RNA


Some examples of aptamer targets material

Small molecules

Zn2

ATP

adenosine

cyclic AMP

GDP

FMN (and an RNA aptamer is found naturally in E.coli)

cocaine

dopamine

amino acids (arginine)

porphyrin

biotin

organic dyes (cibacron blue, malachite green)

neutral disaccharides (cellobiose, and cellulose)

oligopeptides

aminoglycoside antibiotics (tobramycin)

Proteins

thrombin

HIVtat

HIV rev

Factor IX

VEGF

PDGF

ricin

large glycoproteins such as CD4

anthrax spores (?)


Electrostatic surface map: materialred= - blue = +

Base flap shuts door


One anti-Rev aptamer: material

binds peptide in

alpha-helical conformation

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

Another anti-Rev aptamer:

binds peptide in an

extended conformation

MS2 protein as beta sheet bound via protruding side chains


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. Nature419: 90-94.

Reading:

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.

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

( R-3’O-P-O-3’-R-T )


Anti-Factor IX RNA aptamer isolated by SELEX clotting factor IX

Kd for Factor IX = 0.6 nM

F_IXa + F_VIIIa cleaves F_X

Aptamer inhibits this activity

+aptamer-PEG,

Clotting time increase

+aptamer+PEGylation

Mutant version

-aptamer == 1

Conjugate to polyethyleneglycol to increase bloodstream lifetime

PEG = polyethyleneglycol polymer,

appended to decrease clearance rate.


An clotting factor IXantidote 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!

Antidote 5-2 design = the open squares

16-fold excess

Anti-coagulant activity

duplexed

In human plasma

free aptamer

Scrambled

antidote

+Oligomer 5-2

Ratio of anti- to aptamer


Antidote acts fast clotting factor IX

(10 min)

Anti-coagulant activity

Need 10X antidote

Antithrombin aptamer antidote

tested in human serum

Ratio antidote/aptamer

Anti-coagulant activity

Time (min)

Anti-coagulant activity

Antidote lasts a long time

Time (hr)


Reduced clotting clotting factor IX

Reversed by antidote

In serum of patients with

heparin-induced thrombocytopenia

(heparin can no longer be used)


Macugen: an RNA aptamer that binds VEGF and clotting factor IX

is marketed for adult macular degeneration (wet type)

From the label:

R

Inverted ribo-T

3’-3’ to protect 3’ end

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.

VEGF = vascular endothelial growth factor


Ribozymes = RNA enzymes clotting factor IX

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 sequence

GR-binding site

secondary structure

Conserved base analysis (100’s)  confirms structure

X-ray diffraction: a few 3-D structures


lariat clotting factor IX

(natural ribozymes)

Free guanosine

No lariat

lariat

+

+

+


Point of cleavage clotting factor IX

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)


You can use SELEX to isolate new artificial ribozymes clotting factor IX

Tang, J. and Breaker, R.R. 2000. Structural diversity of self-cleaving ribozymes. Proc Natl Acad Sci U S A97: 5784-5789.

1015 DNA molecules with T7 promoter

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

Proposedcleavage zone

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

Now add Mg++

Selecting for cleavage

anywhere in the zone

Isolate the successfully cleaved by size on gels

Proposedcleavage zone

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


New synthetic ribozymes, and DNAzymes clotting factor IX

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”)


Combine an aptamer clotting factor IXand a ribozyme  Allosteric ribozyme

Catalytic activity can be controlled by ligand binding !

Positive or negative.

Modular

Molecular switches, biosensors


Selection of an allosterically clotting factor IXactivated ribozyme

Isolation of aptamer-ribozyme combinations

that respond to ligand binding.

Randomize the “communication module”

Iterations

Select with decreasing activation times for better and better binders.

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.


fluorophore clotting factor IX

quencher

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 Chem9: 2521-2524.

Start with a theophylline-dependent ribozyme:

Analogy:

A molecular “beacon” that respond to nucleic acid

hybridization


+ clotting factor IX

Too short to maintain a

stable duplex structure with SWI 58

Separate substrate molecule (in trans), fluorescently tagged

Nearby

quenching group kept close by hybridization


H clotting factor IX

theophylline

5X over background

caffeine

good specificity

Not so sensitive (0.3 mM)


over spontaneous reaction clotting factor IX

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)


Gold, L. et al., clotting factor IXAptamer-Based Multiplexed Proteomic Technology for

Biomarker Discovery

PLoS ONE, 1 December 2010,

Volume 5, e15004

SomaLogic, Inc.


Some prominent aptamer companies: clotting factor IX

Archemix (Boston) RNA aptamers

Somalogic (Colorado) DNA aptamers

Noxxon (Germany) “spiegelmers”


siRNA = clotting factor IXshort interfering RNA

Double stranded (DS) RNA

miRNA = microRNA

naturally occurring siRNA

Dicer

siRNA:

~22mer, 2 nt overhangs

(Primary transcript)

RISC: RNA-induced

silencing complex

RISC activation

Single-stranded RNA

Protect against viral RNA, repetitive sequence transcripts

More common

Anneals to mRNA target

No cleavage if imperfectly complementary, but translation inhibition

Cleavage if perfectly complementary

mRNA target cleavage and degradation


miRNA = clotting factor IXmicroRNA, naturally occurring siRNA

Primary transcriptpoly- or mono-cistronic

miRNA = microRNA

naturally occurring siRNA

Enzymatic processing

Pre-microRNA

nucleus

cytoplasm

Dicer

Protect against viral RNA, repetitive sequence transcripts

Mature miRNA

Anneals to mRNA target: Cleavage if perfectly complementary

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


Introduction of long DS RNA into clotting factor IXmammalian 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)


Generation of siRNA clotting factor IXin vitro

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

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

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

Introduce perfect hairpin RNA into cells,

let Dicer make siRNA

Introduce imperfect hairpin RNA into cells(based on mRNA sequence) and

let Dicer make miRNA


Limitations of exogenous siRNA treatment for silencing in mammalian cells

Transient nature of the response (~3 days)

Transfection problems (cell type, refractoriness)

Non-renewable nature of siRNAs ($$)


Generation of siRNA mammalian cellsin vivo (can give permanent knockdown)

Not good for interferon-

responsive cells(long DS RNA induces death response)

Allow trans-association

(TTTTT acts as a terminator)

(Pol III)

Most common,

using U6 or H1 promoter

U6 = small nuclear RNA used for splicing.H1 = RNA element of RNase P, used in tRNA processing.

(Pol II)

miRNA


Potential determinants of efficient siRNA-directed gene silencing

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.

Delivery

Transfection (lipofection, electroporation, hydrodynamic injection (mouse))

Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells)


Some applications: silencing

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)

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)


Delivery in an intact organism silencing

Hydrodynamic injection (sudden large volume) of straight siRNA (no vector) into the tail vein of a newborn mouse

Get silencing of co-injected luciferase vector in a variety of tissues

High throughput siRNA for gene discovery

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)


Systemic RNAi: worms, plants, mammals silencing

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

Worms: effect can last though several generations

Amplified by reverse transcriptase

Influx/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”


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 protein

Enough to satisfy the protein requirement of 500 million people.

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


Delta-cadinene synthase tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054

Target the mRNA specifying the first step in gossypol synthesis


Recombinant plasmid T1 grows in the bacteria tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054 Agrobacterium tumefaciens

which can be used as a vector for plant transfection

dCS = delta-cadinene synthase

shRNA

neo gene

terminator

 alpha-globulin promoter

a-globulin promoteris active only in seeds

The T-DNA region of the binary vector pAGP-iHP-dCS. Arrows indicate the primers used in the PCR analyses.

RB-right T-DNA border

tOCS:octopine synthase terminator

dCS: 604-bp d-cadinene synthase sequence

pAGP: cotton a-globulin promoter (seed specific)

pNOS: nopaline synthase promoter

nptII:neomycin phosphotransferase II

tNOS: nopaline synthase terminator

LB: left T-DNA border.


NEO-RESISTANT TRANSFECTANT PLANTS tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054

Ten seeds from two transgenic plants from F1 of selfed matings

0.1 ug/mg

PCR for transgene

Note transgene-null segregants have normal gossypol levels


HPLC (high performance liquid chromatography) tissue-specific reduction of toxic gossypol. Ganesan Sunilkumar*, LeAnne M. Campbell*, Lorraine Puckhaber†, Robert D. Stipanovic†, and Keerti S. Rathore. PNAS (2006) 103: 18054

Null segregant

Spots on seed indicate terpenoid glands


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


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.


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 bean

cassava bean


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