Targeting duplex DNA:

strategies and applications

Maxim Frank-Kamenetskiimfk@bu.edu

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