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Transcript of Targeting duplex DNA: strategies and applications Maxim Frank-Kamenetskii [email protected] Boston...
Targeting duplex DNA:
strategies and applications
Maxim [email protected]
Boston Universityreprints at:
http://www.bu.edu/cab
How can we sequence-
specifically target
the DNA duplex?
H DNATriplex
Triplex Displaced strand
Displaced strand
1985
base triads
Hoogsteen pairing
Watson-Crick pairing
Hoogsteen pairing
Watson-Crick pairing
The DNA double helix
minor groove
major groove
Old-fashioned single-molecule experiment
JMB 1993 JMB 1993
PNA as a tool
for targeting duplex DNA
Peptide Nucleic Acid carries is the same bases as DNA (red), but
has a totally different protein-like backbone (blue)
PNA – A DNA Mimic with Unique Properties
N
N
N
N
NH2
N
N
O
NH2
OO
O
P
O
O O
O
O
P OO
DNA
N
O
NH
O NH
N
N
O
NH2
N
O
O
N
N
N
N
NH2
PNA
Nielsen et al. 1991
PNA features
Neutral backbone
Stronger and faster binding to nucleic acids
Strand invasion into duplex DNA
High sequence-specificity
No peptide no degradation by protease
No nucleic acid no degradation by nucleases
PNA/DNA duplexes are more stable than DNA/DNA duplexes
Peter Lansdorp (University of British Columbia, Canada)
FISH of telomeres using PNA
PNA FISH for bacterial detection
AdvanDx Inc. , Woburn, MA
Staphylococcus aureus (green)
PNA openersTriplex
InvasionDouble Duplex
Invasion
Pseudocomplementary
pcPNA any base
composition
Homopyrimidine PNA
base triads
Hoogsteen pairing
Watson-Crick pairing
Hoogsteen pairing
Watson-Crick pairing
A pair of pseudocomplementary PNAs (pcPNAs) invade into the DNA double helix in a
strictly sequence-specific manner
PNAS 2004
Triplex Invasion into Duplex DNA by PNA “Openers”
PNA “opener”: Two homopyrimidine PNA oligomers connected by a flexible linker (bis-
PNA)
Homopurine site within dsDNAdsDNA
Triplex Invasion Complex
“ P-loop “ H-Lys2-JJTJTJTT
H2N-Lys -CCTCTCTTlinker
Example:
Hoogsteen
pairingWatson-Crick
pairing
J bases eliminate pH dependence of triplex invasion
G
CJ
HN
O
N N
NN
R
H
N N
N
R
O
HH
H
H
HH
N
O
NN
R
H
H
H
J*G:C (pH7)C*G:C (pH5)
Hoogsteen
pairing
CC+
G
H
N
O
N
N+
R
HH
HN
O
N N
NN
R
H
N N
N
R
O
HH
H
H
H
H
H
Watson-Crick
pairing
+
++
Targeting duplex DNA through PD-loop
N=4-10
5' 3'
3' 5'
NH2COOH
PNA openers = 6-10
Two PNA openers are able to sequence-specifically hybridize to complementary target sites in duplex DNA DNA probe can hybridize to the displaced strand forming a stable complex
5' 3'
PD-loop
PNAS 1998
Capturing duplex DNA using PD-loop
Capturing a fragment from the entire yeast
genomePD-loop
PNAS 1998
Applications of PNA openers
............... ............... ............... ..
............
................... ...............
PNA openers and some of their applications
dsDNA
bis-PNA openers
Locally open dsDNA
Hybridization of DNA or PNA beacon
.........
........... ............... ....
Q F
DNA detection
....
.... ........
.........
........... ............... ....
“Earring Probe”
Assembly of novel DNAstructures, DNA diagnostics
Hybridization/circularization of oligonucleotide
....
.... ........
DNA sequencing, Ligand Mapping
............
...................
... .. . ...“Artificial primosome”
Hybridization/extension of primer
... .. . ... ... .. . ...
homopyrimidine sequence
Molecular beacons
JACS 2002
DNA polymerase pausing due to drug binding to dsDNA
Nascent DNA strand
PNA openers
DNA polymerase
ligand
JMB 2003
Mapping drug’s binding sites on dsDNA via artificial primosome
PNA I
PNA II
DNA detection
using PNA openers
PD-loop as a tool
for detection of short signature sites
New methods of DNA-based detection
AEM 2007
BMC 2007
Proof-of-principle studies on bacterial cells
chosen signature sites:
21-nt-target site in E.coli cold shock protein gene
GGAGAGAGACTCAAAAGAAGG
23-nt-target site in B.subtilis the phosphoglycerate dehydrogenase gene
GAAAAGAAACCCTTCAGAGGAAG
22-nt-target site in S.mutans the wall-associated protein gene
AAAAGAGGTATTTTAAGAGGAA
(PNA binding sites are underlined)
These sites are unique for each of the bacteria throughout the Bacterial Genomes Database
E.coli
B.subtilis
S.mutans
AEM 2007
Potential Application:Viral DNA Detection
Clinical specim entaken
DNA isolated & labeled
Barcode generated& analyzed
HSV-1HSV-2
VZV
Solid-state nanopore
NanoLetters 2010
NanoLetters 2010
Duplex DNA labeling using nicking enzymes
NAR 2008
PNA openersTriplex
InvasionDouble Duplex
Invasion
Pseudocomplementary
pcPNA any base
composition
Homopyrimidine PNA
-PNA
ArtDNA 2010
Capturing duplex DNA using PD-loop
Capturing a fragment from the entire yeast
genomePD-loop
PNAS 1998
Affinity capture using -PNA:linear dsDNA
ArtDNA 2010
Affinity capture using -PNA:supercoiled DNA (scDNA)
ArtDNA 2010
Acknowledgements• Boston University
• Irina Smolina
• Heiko Kuhn
• Nancy Miller
• Amit Meller and his group
• Harvard Medical School• Charles Lee
• US Genomics• Katya Protozanova
• Gary Jaworski
• Rhea Mahabir
• Copenhagen University• Peter Nielsen
• Carnegie Mellon University• Danith Ly
• Funding:
• Wallace H. Coulter Foundation.
• NIH