Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes

Abstract

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3′ UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2′-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5′ and 3′ termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3′-side CL improves nuclease resistance, whereas the 5′-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3′ part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Synthesis and Application of Interstrand Cross‐Linked Duplexes by Covalently Linking a Pair of Abasic Sites

“…We also examined the inhibitory activity against the miRNA21 (miR‐21) of CL‐AMO in HeLa cells using the dual luciferase assay (Mie et al., ). As shown in Figure , both anti‐miR‐21 (AS) and AS flanked by single‐stranded 12‐mer (5′3′SS) inhibited little miR‐21 at 48 hr after transfection; however, 5′3′DS flanked by perfect match 12‐mer duplexes (DSs) showed a significant inhibition against miR‐21.…”

“…This protocol describes the evaluation of the inhibitory activity of synthesized crosslinked anti-miRNA oligonucleotides (CL-AMO) against the miRNA by using the dual luciferase assay (Mie et al, 2018). AMOs flanked by CLs are transfected into HeLa cells using Lipofectamine R 2000, and gene silencing activities against miR-21 are evaluated using a dual luciferase assay system.…”

“…We also examined whether the 2′ ‐O‐ methyl RNA (MeRNA) duplex would be cross‐linked at an AP site pair (Mie et al., ). As described in Basic Protocol 3, a 12‐mer MeRNA duplex containing a 2′‐deoxyuridine on each strand was treated by UDG and then cross‐linked with aoNao.…”

“…We developed a site‐specific CL reaction that links a pair of AP sites in a complementary DNA duplex, as described in Basic Protocol 2 (Ichikawa et al., ). Recently, we constructed an AMO flanked by an interstrand cross‐linked 2′‐O‐methylated RNA duplex (CL) and found that the CL affects the anti‐miRNA activity (Mie et al., ).…”

“…We recently developed a sequence-specific cross-linking reaction that conjugates a pair of apurinic/apyrimidinic (AP) sites on complementary oligodeoxynucleotides (ODNs) using a cross-linker containing bis(aminooxy) groups (Ichikawa et al, 2012). The method is also effective for the preparation of cross-linked RNA molecules, and we applied it to the construction of bioactive anti-miRNA oligonucleotides (AMOs) flanked by interstrand cross-linked 2 -O-methyled RNA at the 5 -and 3 -termini (Mie et al, 2018). Figure 1 shows a schematic of the cross-linking reaction of the duplex DNA described here.…”