Optimizing anti-gene oligonucleotide 'Zorro-LNA' for improved strand invasion into duplex DNA.
Zorro-LNA (Zorro) is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the potential of specific binding to each strand of duplex DNA. The first-generation Zorros are formed by two hybridized LNA/DNA mixmers (2-ON Zorros) and was hypothesized to strand invade. We have now established a method, which conclusively demonstrates that an LNA ON can strand invade into duplex DNA. To make Zorros smaller in size and easier to design, we synthesized 3'-5'-5'-3' single-stranded Zorro-LNA (ssZorro) by using both 3'- and 5'-phosphoramidites. With ssZorro, a significantly greater extent and rate of double-strand invasion (DSI) was obtained than with conventional 2-ON Zorros. Introducing hydrophilic PEG-linkers connecting the two strands did not significantly change the rate or extent of DSI as compared to ssZorro with a nucleotide-based linker, while the longest alkyl-chain linker tested (36 carbons) resulted in a very slow DSI. The shortest alkyl-chain linker (3 carbons) did not reduce the extent of DSI of ssZorro, but significantly decreased the DSI rate. Collectively, ssZorro is smaller in size, easier to design and more efficient than conventional 2-ON Zorro in inducing DSI. Analysis of the chemical composition of the linker suggests that it could be of importance for future therapeutic considerations.
Zaghloul
Eman M
EM
Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Stockholm, Sweden. eman.zaghloul@ki.se
Madsen
Andreas S
AS
Moreno
Pedro M D
PM
Oprea
Iulian I
II
El-Andaloussi
Samir
S
Bestas
Burcu
B
Gupta
Pankaj
P
Pedersen
Erik B
EB
Lundin
Karin E
KE
Wengel
Jesper
J
Smith
C I Edvard
CI
D004247:DNA / Q000737:chemistry*; D020868:Gene Silencing; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; C477371:locked nucleic acid; C051802:triplex DNA; D004247:DNA