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![Page 1: Targeting duplex DNA: strategies and applications Maxim Frank-Kamenetskii mfk@bu.edu Boston University reprints at: .](https://reader035.fdocuments.net/reader035/viewer/2022062322/56649d0a5503460f949dc6cf/html5/thumbnails/1.jpg)
Targeting duplex DNA:
strategies and applications
Maxim [email protected]
Boston Universityreprints at:
http://www.bu.edu/cab
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How can we sequence-
specifically target
the DNA duplex?
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H DNATriplex
Triplex Displaced strand
Displaced strand
1985
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base triads
Hoogsteen pairing
Watson-Crick pairing
Hoogsteen pairing
Watson-Crick pairing
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The DNA double helix
minor groove
major groove
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Old-fashioned single-molecule experiment
JMB 1993 JMB 1993
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PNA as a tool
for targeting duplex DNA
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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
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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
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PNA/DNA duplexes are more stable than DNA/DNA duplexes
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Peter Lansdorp (University of British Columbia, Canada)
FISH of telomeres using PNA
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PNA FISH for bacterial detection
AdvanDx Inc. , Woburn, MA
Staphylococcus aureus (green)
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PNA openersTriplex
InvasionDouble Duplex
Invasion
Pseudocomplementary
pcPNA any base
composition
Homopyrimidine PNA
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base triads
Hoogsteen pairing
Watson-Crick pairing
Hoogsteen pairing
Watson-Crick pairing
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A pair of pseudocomplementary PNAs (pcPNAs) invade into the DNA double helix in a
strictly sequence-specific manner
PNAS 2004
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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
+
++
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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
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Capturing duplex DNA using PD-loop
Capturing a fragment from the entire yeast
genomePD-loop
PNAS 1998
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Applications of PNA openers
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............... ............... ............... ..
............
................... ...............
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
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Molecular beacons
JACS 2002
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DNA polymerase pausing due to drug binding to dsDNA
Nascent DNA strand
PNA openers
DNA polymerase
ligand
JMB 2003
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Mapping drug’s binding sites on dsDNA via artificial primosome
PNA I
PNA II
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DNA detection
using PNA openers
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PD-loop as a tool
for detection of short signature sites
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New methods of DNA-based detection
AEM 2007
BMC 2007
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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
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Potential Application:Viral DNA Detection
Clinical specim entaken
DNA isolated & labeled
Barcode generated& analyzed
HSV-1HSV-2
VZV
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Solid-state nanopore
NanoLetters 2010
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NanoLetters 2010
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Duplex DNA labeling using nicking enzymes
NAR 2008
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PNA openersTriplex
InvasionDouble Duplex
Invasion
Pseudocomplementary
pcPNA any base
composition
Homopyrimidine PNA
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-PNA
ArtDNA 2010
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Capturing duplex DNA using PD-loop
Capturing a fragment from the entire yeast
genomePD-loop
PNAS 1998
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Affinity capture using -PNA:linear dsDNA
ArtDNA 2010
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Affinity capture using -PNA:supercoiled DNA (scDNA)
ArtDNA 2010
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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