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.
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.
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:
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).
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
Ligand is immobilized here.
Small molecule or large molecule.
Some examples of aptamer targets material
FMN (and an RNA aptamer is found naturally in E.coli)
amino acids (arginine)
organic dyes (cibacron blue, malachite green)
neutral disaccharides (cellobiose, and cellulose)
aminoglycoside antibiotics (tobramycin)
large glycoproteins such as CD4
anthrax spores (?)
Electrostatic surface map: materialred= - blue = +
Base flap shuts door
One anti-Rev aptamer: material
binds peptide in
Hermann, T. and Patel, D.J.2000. Adaptive recognition by nucleic acid aptamers. Science287: 820-825.
Another anti-Rev aptamer:
binds peptide in an
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.
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
Clotting time increase
-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
In human plasma
Ratio of anti- to aptamer
Antidote acts fast clotting factor IX
Need 10X antidote
Antithrombin aptamer antidote
tested in human serum
Antidote lasts a long time
Reduced clotting clotting factor IX
Reversed by antidote
In serum of patients with
(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:
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
Conserved base analysis (100’s) confirms structure
X-ray diffraction: a few 3-D structures
lariat clotting factor IX
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++)
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
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.
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”
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
Using an allosteric ribozyme to create a chemical sensor
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:
A molecular “beacon” that respond to nucleic acid
+ clotting factor IX
Too short to maintain a
stable duplex structure with SWI 58
Separate substrate molecule (in trans), fluorescently tagged
quenching group kept close by hybridization
H clotting factor IX
5X over background
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
Gold, L. et al., clotting factor IXAptamer-Based Multiplexed Proteomic Technology for
PLoS ONE, 1 December 2010,
Volume 5, e15004
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
~22mer, 2 nt overhangs
Protect against viral RNA, repetitive sequence transcripts
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
Protect against viral RNA, repetitive sequence transcripts
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)
(TTTTT acts as a terminator)
using U6 or H1 promoter
U6 = small nuclear RNA used for splicing.H1 = RNA element of RNase P, used in tRNA processing.
Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC.
Base-pairing with mRNA.
Cleavage of 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.
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)
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.
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
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
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
Spots on seed indicate terpenoid glands
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.