Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA.
In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.
Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, 141 86 Huddinge, Stockholm, Sweden.
Pedro M D
PM
Moreno
Sylvain
S
Geny
Y Vladimir
YV
Pabon
Helen
H
Bergquist
Eman M
EM
Zaghloul
Cristina S J
CS
Rocha
Iulian I
II
Oprea
Burcu
B
Bestas
Samir El
SE
Andaloussi
Per T
PT
Jørgensen
Erik B
EB
Pedersen
Karin E
KE
Lundin
Rula
R
Zain
Jesper
J
Wengel
C I Edvard
CI
Smith
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002021:Buffers; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004278:DNA, Superhelical / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry; D044366:Transition Temperature
D002021:Buffers; D004278:DNA, Superhelical; D009841:Oligonucleotides; C477371:locked nucleic acid; C051802:triplex DNA; D004247:DNA; D004262:DNA Restriction Enzymes