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Class 27_11 last updated 12/14/11 12:30 PM
Limitations of siRNA silencing in mammalian cells
Transient nature of the response (~3 days)
Transfection problems (cell type, refractoriness)
Can be cell type specific
Non-renewable nature of siRNAs ($$)
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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 of shDNA, esp. lentivirus (e.g., retrovirus like HIV that can integrate into
non-dividing cells)
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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)
Cells transformed with
tsSV40LTag, which inactivates
p53 at 32o but not at 39o.
Infect with Hu shRNA lentivirus
shRNA library, select cells that
grow at 39o.
Knocked down genes = those
necessary for p53-induced
growth arrest.
NATURE 428. 2004. p. 431
32o
Control
39o
Control
39o
p16K.D.
39o
39o
LTag=large T
antigen
p53K.D. p533+p16K,K.D.
Identified
shRNAs
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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 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”
Discovery of RNA interference using double-stranded RNA
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Nature (1998) 391: 806
Discovered RNAi as they tracked down the effective agent in antisense experiments
(DS RNA contaminating their SS antisense preparations had all the inhibitory activity)
Paper characterized by nice controls and variations:
Several genes, whole animal phenotype, protein product (GFP), RNA level (in situs)
Phenotype of null mutant is specifically mimicked.
Introns and promoter sequences ineffective.
DS RNA from a different sequence + SS antisense RNA vs. the target: ineffective
DS RNA linked (chimeric molecule) to a single stranded portion vs, the target: ineffective
Transport of DS RNA between cells and amplification implied.
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In situ hybridizations
No probe
No RNA injected
SS
antisense
RNA
DS RNA
Transcript disappears (RNA degraded)
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Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral
Response in C. elegans. Oded Rechavi , Gregory Minevich, Oliver
Hobert Cell 147, 1248, Dec. 11, 2011
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.
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Who’s binding whom?
Protein:
thrombin
(blood protease)
thrombin-binding
RNA aptamer
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SELEX: Systematic Evolution of Ligands by Exponential Enrichment
E.g.:
Have a random 40-mer synthesized,
centered between 2 arbitrary 20-mers (PCR sites)
20-mer
Random 40
20-mer
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 found in the human genome.
These 1015 sequences are called “sequence space” being examined here.
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
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 particular
target of interest (protein, small molecule).
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SELEX: Systematic Evolution of Ligands by Exponential Enrichment . . . for RNA (or DNA)
DNA
(1015)
RNA
RNA
Essential elements:
1) Synthesis of randomized DNA
sequences
2) In vitro T7-mediated RNA
synthesis from DNA
3) Affinity chromatography
4) RT-PCR
Ligand is
immobilized here.
Small molecule
or large molecule
DNA, add T7 promoter
to one pcr primer
RNA
RNA
e.g., the soluble form of the
immobilized affinity column material
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Some examples of aptamer targets
Small molecules
Zn+2
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
HIV tat
HIV rev
Factor IX (clotting factor)
VEGF
PDGF
ricin
large glycoproteins such as CD4
anthrax spores (?)
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Table 3 Therapeutic Aptamers in Clinical Use or Clinical Development (Ph1 or 2) as of
June 2009
Therapeutic
Target
Disease Indication
Route/Method
Administra
tion
Macugen™
(Pegaptanib)
VEGF
Macular degeneration
Intravitreal
ARC1779
von Willebrand
factor
REG1
Compound ID
Thrombotic
microangiopathy;
Adjunct to carotid
endarterectomy
Intravenous
infusion
Factor IXa
Coronary artery bypass;
Percutaneous
coronary intervention
Intravenous
bolus
AS1411
Nucleolin
Acute myeologenous
leukemia; Renal cell
carcinoma
Intravenous
infusion
E10030
PDGF-b
Macular degeneration
Intravitreal
ARC1905
Complement
factor 5
Macular degeneration
Intravitreal
NU172
Thrombin
Coronary artery bypass
Intravenous
infusion
Tobramycin (aminoglycosde antibiotic)
RNA aptamer
Base flap shuts door
Electrostatic
surface map:
red= - blue = +
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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 an
extended conformation
MS2 protein as beta
sheet bound via
protruding A.A. side
chains
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Therapeutic use of an aptamer that binds to and inhibits clotting factor IX
Reading: 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.
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 angioplasty is
heparin, which has some toxicity and is difficult to
control.
Inverted T at 3’ end (3’-3’)
slows exonucleolytic
degradation
( R-3’O-P-O-3’-R-T )
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Anti-Factor IX RNA aptamer isolated by SELEX
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Kd for Factor IX = 0.6 nM
F_IXa + F_VIIIa cleaves F_X
4 nM 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.
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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!
aptamer antidote
complex
Antidote 5-2
design = the
open squares
In human plasma
+Oligomer 5-2
Anti-coagulant
activity
16-fold excess
antidote
duplexed
free aptamer
Scrambled
antidote
Ratio of anti- to aptamer
Other antidotes
Anti-coagulant activity
Anti-coagulant activity
Anti-coagulant activity
Antithrombin
aptamer antidote
tested in human
serum
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Need 10X antidote
Ratio antidote/aptamer
Antidote acts fast
(10 min)
Time (min)
Antidote lasts a long time
Time (hr)
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Macugen: an RNA aptamer that binds VEGF and
is marketed for adult macular degeneration (wet type)
From the label:
R
Where R is
and contains a PEG chain of ~ 900 ethylene glycol units.
Inverted ribo-T
3’-3’ to protect
3’ end
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[ωmethoxypoly(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
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BioTechniques 51:413-416 (December 2011) doi 10.2144/000113786
Before selection for binding to immobilized thrombin
After selection
Biacore analysis of thrombin binding
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Ribozymes = RNA enzymes
1982 Tom Cech: Tetrahymena rRNA intron is self-spliced out
(requires 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
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(natural ribozymes)
Free
guanosine
lariat
No lariat
+
lariat
+
+
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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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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++)
Proposed
cleavage zone
RT -> cDNA: Cleavage
zone is rebuilt by being
part of the primer.
Now add Mg++
Selecting for
cleavage
anywhere in the
zone
Isolate those
successfully
cleaved by size on
gels
Proposed
cleavage zone
i.e., al 16 dinucleotides
present as possible cleavage
sites
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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
Selection of an allosterically activated ribozyme
Isolation of aptamer-ribozyme combinations
that respond to ligand binding.
Randomize the “communication module”
FMN=flavin mononucleptide
Iterations
Select with decreasing activation times for better and better binders.
And:
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.
Catalytic 29
domain
(ribozyme)
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
fluorophore
quencher
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+
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
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H
theophylline
5X over background
caffeine
good specificity
But not so sensitive
(0.3 mM)
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Some DNAzyme activities
Compare protein enzymes,
Typically 6000 on this scale
(100/sec)
Emilsson, G. M. and R. R. Breaker (2002).
Deoxyribozymes: new activities and
new applications.
Cell Mol Life Sci 59(4): 596-607.
over spontaneous reaction
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Wash;
Photorelease
DNA aptamer
(+fluorescent
tag)
Nonspecific
Bind
complexes
to avidin
beads
Biotinylate
bound
proteins
Capture
again on
avidin
beads
Gold, L. et al.,
Aptamer-Based
Multiplexed Proteomic
Technology for
Biomarker Discovery
PLoS ONE, 2010,
5, e15004
SomaLogic, Inc.
Release DNA
aptamers with
high pH and
collect
Identify and quantify release
aptamer DNA on a
microarray using
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Some prominent aptamer companies:
Archemix (Boston) RNA aptamers
Somalogic (Colorado) DNA aptamers
Noxxon (Germany) “spiegelmers”
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• Got this far
• Material beyond this point will not be
covered on the final exam.
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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 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.
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Delta-cadinene synthase
Target the mRNA specifying the first step in gossypol synthesis
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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
 alphaglobulin
promoter
a-globulin promoter is 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.
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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
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HPLC (high performance liquid chromatography)
Null segregant
Spots on seed indicate terpenoid glands
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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
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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 = betaN-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