Expanding the design horizon of antisense oligonucleotides with alpha-L-LNA.
Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. In this work, two different diastereoisomers of LNA were studied: the beta-D-LNA and the alpha-L-LNA (abbreviated as beta-D-LNA and alpha-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing beta-D-LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA-DNA-LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif-dependent, and requires the right balance between gap size and affinity. Compared to beta-D-LNA, alpha-L-LNA shows superior stability against a 3'-exonuclease. The design possibilities of alpha-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of alpha-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, alpha-L-LNA and beta-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with alpha-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with beta-D-LNA results in a drop in antisense potency. Our findings indicate that alpha-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.
Santaris Pharma A/S, Bøge Allé 3, DK-2970 Hørsholm, Denmark.
Miriam
M
Frieden
Signe M
SM
Christensen
Nikolaj D
ND
Mikkelsen
Christoph
C
Rosenbohm
Charlotte A
CA
Thrue
Majken
M
Westergaard
Henrik F
HF
Hansen
Henrik
H
Ørum
Troels
T
Koch
D001483:Base Sequence; D015536:Down-Regulation; D005092:Exonucleases / Q000378:metabolism; D005818:Genetic Engineering; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013237:Stereoisomerism; D013696:Temperature
D020319:Oligodeoxyribonucleotides, Antisense; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D008156:Luciferases; D005092:Exonucleases; C022650:spleen exonuclease; D016914:Ribonuclease H; D015719:Single-Strand Specific DNA and RNA Endonucleases