Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs).

Pawan Kumar,Tom Brown,Ysobel Ruth Baker,Lynda Truong,Afaf Helmy El-Sagheer

doi:10.1021/acsomega.8b01086

Abstract

Dinucleoside phosphoramidites containing a triazole internucleotide linkage flanked by locked nucleic acid (LNA) were synthesized and incorporated into oligonucleotides (ONs). ONs bearing both LNA and triazole at multiple sites were obtained and their biophysical properties including enzymatic stability and binding affinity for RNA and DNA targets were studied. t-LNAs with four incorporations of a dinucleoside monomer having LNA on either side of the triazole linkage bind to their RNA target with significantly higher affinity and greater specificity than unmodified oligonucleotides, and are remarkably stable to nuclease degradation. A similar but reduced effect on enzymatic stability and binding affinity was noted for LNA only on the 3′-side of the triazole linkage. Thus, by combining unnatural triazole linkages and LNA in one unit (t-LNA), we produced a promising class of ONs with reduced anionic charge and potential for antisense applications.

Highlights

Antisense oligonucleotides (ASO) are short single-stranded nucleic acids that bind to their RNA target in a sequence-specific manner and modulate translation or RNA splicing.[1]
The efficient synthesis of dinucleoside phosphoramidites containing a triazole linkage and locked nucleic acid (LNA) sugars has been achieved. Access to these dinucleoside phosphoramidites allowed the incorporation of the triazole linkage flanked by LNA at multiple positions in oligonucleotides, including the central region and the 3′- and 5′-ends
There are several examples of a short triazole linkage being introduced into oligonucleotides via phosphoramidite monomers, but in all the cases, the duplexes were greatly destabilized.[21−23] There is one report of this short triazole being combined with LNA and added to oligonucleotides by phophoramidite chemistry, but unlike our triazole, this particular linkage was strongly duplexdestabilizing despite the presence of LNA.[15]

Summary

■ INTRODUCTION

Antisense oligonucleotides (ASO) are short single-stranded nucleic acids (typically 15−25 nucleotides in length) that bind to their RNA target in a sequence-specific manner and modulate translation (protein synthesis) or RNA splicing.[1]. ONs bearing monomers W or Y at the 5′-end bind to their RNA targets with a slightly lower affinity than the unmodified DNA strand (compare ON2U with ON2W and ON2Y, ΔTm of −2.5 and −3.3 °C, respectively). The extreme stability of ON6Z to snake venom phosphodiesterase shows that there is a synergistic effect between LNA and triazole, justifying the modified DNA linkage design on which this study is based. T-LNAs with LNA on either side of the triazole linkage have the highest stability against nucleolytic degradation, in addition to the strongest affinity for RNA targets

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES