Synthesis And Property Of 2’-deoxy-2’-fluorine-4’-substitued Arabinouridine Modified Antisense Oligonucleotide

RNA as the drug target, there is a broad developing prospect for antisense oligonucleotide drugs in the treatment of various major diseases, such as viral infectious disease and cancer, and the key to improve the druggability of the oligonucleotide molecule is proper chemical modification. In order to develop novel chemical structures for nucleic acids, we proposed a new chemical modification strategy——2’-deoxy-2’-fluorine-4’-substitued-arabinouridine(2’-F-4’-X-ara U) based on the unique properties of 2’-deoxy-2’-fluorine-β-D-arabinonucleotide(2’-F-ANA). Starting with 2’-F-ara U, the strategy was anticipated to enhance nuclease resistance, increase target affinity and specificity, improve biological activity and pharmacokinetic properties, and reduce toxicity by introducing different substituents into sugar 4’ position.With hydroxymethyl group(-HM) and methoxy group(OMe) at the 4’ position of 2’-F-ara U separately, four monomers were designed, synthesized and successfully incorporated into the specific sequence by the solid phase phosphoramidite method. Then the properties of modified oligonucleotides were evaluated with respect to thermodynamic stability(affinity and specificity), nucleic acid secondary conformation and enzyme stability, and the influence of fluorine atom on the corresponding properties was also investigated at the same time. The results are as follows:(1) 2’-F-4’-HM-ara U modification with normal phosphate backbone has no effect on the binding affinity with ss RNA or ss DNA, and the modified ON can still form stable double helix with its complementary sequence. While the abnormal backbone modification has a great impact on the double-stranded binding capacity, which is not appropriate for further study. Additionally, 2’-F-4’-HM-ara U modification can significantly increase the mismatch recognition ability of ONs towards G base(especially for the r G), thus having a relatively better binding specificity towards the matched base A compared with natural T and 2’-F-ara U monomer. Moreover, 2’-F-4’-HM-ara U modification can notably enhance the nuclease stability of ONs towards SVPDE, and the correlation of the effect of 2’substituent on enzyme resistance and that of 4’substituent requires a further investigation.(2) Compared with the reference strand(unmodified and 2’-F-ara U modified), oligonucleotides containing 2’-F-4’-OMe-ara U modification can form stable double strands towards ss DNA or ss RNA and exhibit obvious RNA selectivity when hybridizing with its complementary sequence; however 2’-F-4’-OMe-r U modification doesn’t have such RNA selectivity. Besides, 2’-F-4’-OMe-ara U modification show better binding specificity towards ss RNA than ss DNA, while the 2’-F-4’-OMe-r U modification has similar good mismatch recognition ability towards both ss DNA and ss RNA. The foregoing discussion suggests that the orientation of 2’F atom does have a notable influence on the hybridization properties of oligonucleotides, that is, 2’βF seems more beneficial to RNA selective hybridization ability and better binding specificity towards ss RNA other than 2’αF. What’s more, 2’-F-4’-OMe-ara U modification can considerably improve the nuclease hydrolytic stability, and 2’βF is more favorable to increase the enzyme resistance when compared with 2’αF.