Free-radical ring closure to conformationally locked α-L-carba-LNAs and synthesis of their oligos: nuclease stability, target RNA specificity, and elicitation of RNase H.
A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the β-D-LNA-T or β-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.
Li
Qing
Q
Program of Bioorganic Chemistry, Institution of Cell and Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden.
Yuan
Fengfeng
F
Zhou
Chuanzheng
C
Plashkevych
Oleksandr
O
Chattopadhyaya
Jyoti
J
D003500:Cyclization; D005609:Free Radicals / Q000737:chemistry; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000097:blood / Q000138:chemical synthesis* / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000097:blood / Q000138:chemical synthesis* / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism
D005609:Free Radicals; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D016914:Ribonuclease H