Download Class 26 - Columbia University

Class 26_2011 updated Dec. 8, 201 12:45 AM
Recombinase
Polymerase
Amplification
Radhika Pradhan
Aidan Quinn
Ziwei Song
TwistDx Ltd.
qPCR Primer Design
Portable real-time fluorometer
ADVANTAGES AND
POTENTIAL APPLICATIONS
Advantages
• From 1 molecule DNA or 10 molecules
RNA to detectable levels (billions or
trillions) in 5-10 min
• Low cost and simple reagents means
practical applications are enormous
• Multiplexing allows simultaneous detection
of multiple targets
Potential applications
• Medical diagnosis
– Rapid MRSA
detection
– Microorganism
identification
• Agriculture
– Portable animal
health check
• Biodefense
– Biohazards (anthrax)
• Travel & Public health
– SARS
• Industrial applications
– Food industry
• Classrooms
• Basement labs
• …
Class 26_2011 updated Dec. 8, 201 12:45 AM
Therapeutic intervention at the level of pre-mRNA splicing
A. Interfere with improper splicing caused by splice site creation or activation
E.g., beta-thalassemia (R. Kole) in which a splice site has been created by a mutation
in a hemoglobin gene
Use complementary DNA or RNA (antisense)
Natural DNA/RNA rapidly degraded:
Use modified bases, sugars: PNA, morpholino, 2’ OMe,
Normally, DNA-RNA hybrids + endogenous RNase H type activity RNA destruction
Modified antisense DNA circumvents this problem
(don’t want mRNA destroyed here, want to correct its splicing.)
PNA = peptide nucleic acids
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B. Bias alternative splicing ratios
Target the unwanted isoform exon-intron joint.
e.g., BCL-2 isoforms, one is pro-apoptotic, one anti-apoptotic.
The latter is increased in many cancers
Target the anti-apoptotic isoform in cancer cells.
e.g., GABA-a-gamma-2 receptor
(GABA = gamma amino butyric acid, a neurotransmitter)
Long and short forms. Long form associated with mental illness.
C. Skip offensive exons
e.g., nonsense truncations in dystrophin --->
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Splicing as a target for disease therapy
Nonsense mutation truncates protein
Antisense-induced skipping
x
Expendable exon (e.g., protein with many
repeated domains)
Exon must be multiple of 3 in length to maintain
reading frame after skipping
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RNA modification
Deoxy, or also can
add 2’ MOE
-O-CH2-CH2-O-CH3
MOE = methoxyethyl -
Phosphorothioate deoxyoligonucleotides
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RNA modification for stabilization
ase
Morpholino instead of
deoxyribose or ribose
Modified phosphate
ase
Still base pairs OK!
Even more extreme and more stable: peptide nucleic acids (PNAs)
RNA modification
B = a nucleic acid base
Amide bonds,
No ribose
PNA = peptide nucleic acid
Attached 1 to 4 lysines here
Base pairs even better than natural nucleic acids (higher melting temperatures)
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A. Interfere with improper splicing caused by splice site creation or activation
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Sazani P, et al. and Kole R.
Systemically delivered antisense oligomers upregulate gene expression in mouse tissues
Nat Biotechnol. 2002 Dec;20(12):1228-33.
EGFP: Enhanced green fluorescent protein = model system
Antisense “RNA” injected into tail vein, RNA was modified for stability
Mutant globin intron has activated splice sites
Actin promoter, universally expressed.
Induced exon skipping yields green fluorescence
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No
antisense:
Antisense treatment in
cell cultures (ex vivo) from the
mouse with the mutant
EGFP gene
Control oligo (C)
(50 nt downstream)
was ineffective.
Max. effect = 40%
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C. Skip
offensive
exons
Dystrophin gene 2400 kb, mRNA = 14 kb, 79 exons: a giant gene
Protein maintains muscle cell membrane integrity
Mutation: Duchenne’s muscular dystrophy
Some cases (~half) are due to stop codons (nonsense) in a repetitious exon
(spectrin-like repeat, length = a multiple of 3)
Deliver antisense to the ends of exon with the nonsense mutation in mdx mice (model
for Duchenne’s) to promote the skipping of the nonsense-bearing exon and so avoid
truncation of the protein .
Use AAV (adeno-associated virus) to deliver the antisense gene
Measure:
mRNA with skipped exon
dystrophin protein
muscle histochemistry for dystrophin
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Use antisense RNA to target the branch point upstream of the offending exon 23 and the
donor splice site downstream of the exon.
protein
mRNA
= 3 X 71
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BP = branch point; SD = splice donor
Branch site (consensus
= YNYTRAY)
Sequences targeted by antisense
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U7 promoter
Double target synergistic (loop?) (Kole)
compl. to splice donor site
compl. to branch
ITR = inverted terminal repeat, characteristic of AAV
Consensus binding site
for Sm proteins (to target to
pre-mRNA)
Expression of U7
antisense construct
RT-PCR
transgenic U7SmOPT-A.S.
U7
Endog. U7
0
2
4
6
(slow onset =
conclude slow
mRNA turnover)
13 weeks
included
Splicing assay
(RT-PCR)
Skip exon 23,
after 2-4 wks.
0normal 2
4
6
8
13 weeks
Dystrophin protein
(Western)
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Muscle
immunohistochemistry
Normal
intriguing
Untreated
mdx
Treated
mdx
Top,
middle ,and
bottom
dystrophin
dystrophin-associated antigens
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RNAi = RNA interference
Short double stranded RNA molecules trigger the
degradation of the complementary sequence in the cell,
and can inhibit translation of the targeted mRNA
Their introduction into a cell can greatly reduce any protein whose mRNA
is targeted.
Inhibition is usually incomplete in mammalian cells, but can be considerable (>90%)
Thus “gene knockdown” as opposed to knock-out
siRNA = small inhibitory RNAs
shRNA = short hairpin RNAs (both strands can be coded by one DNA)
asRNA = antisense RNA
miRNA = microRNAs
ncRNA = noncoding RNA
Alternative technologies:
Antisense RNA: block translation or splicing
Ribozymes: RNAs that cleave other RNAs, sequence specifically
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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 RNAactivated), 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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miRNA synthesis and maturation
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mRNA degradation
Inhibits translation of an mRNA
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Generation of siRNA in vitro
Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer)
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
Also, can use controlled RNase to generate
fragments (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
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Generation
of si RNA
in vivo
TRE
Tet-inducible promoter
rtTA3
Reverse tet-transactivator
UBC promoter
Drives expression of rtTA3 and IRES-puro
cPPT
Central Polypurine tract. Helps translocation into nucleus of non-dividing cells
WRE
Enhances the stability and translation of transcripts
turbo RFP
Marker to track inducible shRNAmir expression
Puror
Mammalian selectable marker
AMPr
Ampicillin bacterial selectable marker
5'LTR
5' long terminal repeat
pUC ori
High copy replication and maintenance in e.coli
SIN-LTR
3' Self inactivating long terminal repeat
IRES
Internal ribosome entry site
ZEOr
Bacterial selectable marker
Got this far
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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 (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)
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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 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 (as of 2005)
(e.g., fat metabolism phenotypes found)
Identify the genes affected from sequnce of the siRNA
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NATURE 428. 2004. p. 431
tsSV40LTag 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 p53induced growth arrest.
32o
Control
39o
Control
39o
p16K.D.
39o
39o
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: ineffect
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)
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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SELEX
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 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
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).
Who’s binding whom?
Protein:
thrombin
(blood protease)
RNA thrombinbinding aptamer
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SELEX: Systematic Evolution of Ligands by Exponential Enrichment . . . for RNA (or DNA)
DNA
(1015)
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
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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Tobramycin
Electrostatic
surface map:
red= - blue = +
Base flap shuts door
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!
Antidote 5-2
design = the
open squares
In human plasma
+Oligomer 5-2
Anti-coagulant
activity
16-fold excess
duplexed
free aptamer
Scrambled
antidote
Ratio of anti- to aptamer
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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Reduced clotting
Reversed by antidote
In serum of patients with
heparin-induced thrombocytopenia
(heparin can no longer be used)
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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 ~ 450 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