Nucleic acid aptamers

1
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 its 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.
2
SELEX 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-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).
3
SELEX Systematic Evolution of Ligands by
Exponential. Enrichment for RNA (or DNA)
Essential elements1) Synthesis of randomized
DNA sequences 2) In vitro T7 mediated RNA
synthesis from DNA 3) Affinity chromatography 4)
RTPCR
(1015)
DNA
RNA
Ligand is immobilized here. Small molecule or
large molecule.
DNA
RNA
RNA
4
Some examples of aptamer targets 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 amino
glycoside antibiotics (tobramycin)
Proteins thrombin HIVtat HIV rev Factor
IX VEGF PDGF ricin large glycoproteins such as
CD4 anthrax spores (?)
5
Electrostatic surface mapred - blue
Base flap shuts door
6
Hermann, T. and Patel, D.J.2000. Adaptive
recognition by nucleic acid aptamers. Science
287 820-825.
Another anti-Rev aptamer binds peptide in
an extended conformation
MS2 protein as beta sheet bound via protruding
side chains
7
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. Nature
419 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-3O-P-O-3-R-T )
8
Anti-Factor IX RNA aptamer isolated by SELEX
Kd for Factor IX 0.6 nM
F_IXa F_VIIIa cleaves F_X
Aptamer inhibits this activity
aptamer-PEG,
Clotting time increase
aptamerPEGylation
Mutant version
-aptamer 1
Conjugate to polyethyleneglycol to increase
bloodstream lifetime
PEG polyethyleneglycol polymer, appended to
decrease clearance rate.
9
An 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!
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
10
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)
11
Reduced clotting
Reversed by antidote
In serum of patients with heparin-induced
thrombocytopenia (heparin can no longer be used)
12
Macugen an RNA aptamer that binds VEGF and 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'-d
eoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)C-Gm-(3'?3')-
dT), 5'-ester with a,a'-4,12-dioxo-6-5-(phosph
oonoxy)pentylaminocarbonyl-3,13-dioxa-5,11-diaz
a-1,15-pentadecanediylbis?- methoxypoly(oxy-1,2-
ethanediyl), sodium salt. The molecular formula
for pegaptanib sodium is C294H342F13N107Na28O188P2
8C2H4On (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 67.
VEGF vascular endothelial growth factor
13
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 (100s)
revealed required attributes Internal guide
sequence GR-binding site secondary
structure Conserved base analysis (100s) ?
confirms structure X-ray diffraction a few 3-D
structures
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(natural ribozymes)
Free guanosine
No lariat
lariat



15
Point of cleavage
Hammerhead ribozyme(RNase) can cleave in
cis (hammer head is upside down)
Synthetic variationcleaves in trans
You are in charge of what it will cleave(you
fill in the Ns)
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You can use SELEX to isolate new artificial
ribozymes
Tang, J. and Breaker, R.R. 2000. Structural
diversity of self-cleaving ribozymes. Proc Natl
Acad Sci U S A 97 5784-5789.
1015 DNA molecules with T7 promoter
Keep molecules under non-permissive conditions
so they stay intact (without Mg)
Proposedcleavage zone
RT -gt 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
17
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 transformationsRNase, 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)
18
Combine an aptamer and a ribozyme ?
Allosteric ribozyme Catalytic activity can be
controlled by ligand binding ! Positive or
negative. Modular Molecular switches, biosensors
19
Selection of an allosterically activated 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 A 96 3584-3589.
20
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 Chem 9 2521-2524.
Start with a theophylline-dependent ribozyme
Analogy A molecular beacon that respond to
nucleic acid hybridization
21

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
22
H
theophylline
5X over background
caffeine
good specificity
Not so sensitive (0.3 mM)
23
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
Compare protein enzymes, Typically 6000 on this
scale (100/sec)
24
Gold, L. et al., Aptamer-Based Multiplexed
Proteomic Technology for Biomarker Discovery
PLoS ONE, 1 December 2010, Volume 5, e15004
SomaLogic, Inc.
25
Some prominent aptamer companies Archemix
(Boston) RNA aptamers Somalogic (Colorado) DNA
aptamers Noxxon (Germany) spiegelmers
26
siRNA short 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
27
miRNA microRNA, 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)
28
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(lt30
bp)
29
Generation of siRNA in 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
30
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 ()
31
Generation of siRNA in 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
32
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)
33
Some applications
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)
34
Delivery in an intact organism
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)
35
Systemic RNAi worms, plants, mammals In
plants, get permanent post-transcriptional gene
silencing (PTGS, transcriptional level) Worms
effect can last though several generations Amplifi
ed by reverse transcriptase Influx/efflux via a
specific transmembrane protein (in worms)
Raisons detre? 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
36
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.
37
Delta-cadinene synthase
Target the mRNA specifying the first step in
gossypol synthesis
38
Recombinant plasmid T1 grows in the bacteria
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 tOCSoctopine synthase terminator dCS
604-bp d-cadinene synthase sequence pAGP cotton
a-globulin promoter (seed specific) pNOS
nopaline synthase promoter nptIIneomycin
phosphotransferase II tNOS nopaline synthase
terminator LB left T-DNA border.
39
NEO-RESISTANT TRANSFECTANT PLANTS
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
40
HPLC (high performance liquid chromatography)
Null segregant
Spots on seed indicate terpenoid glands
41
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.
43
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