Synthesis of native-like crosslinked duplex RNA and study of its properties

Abstract

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

Keywords: 6-Vinylpurine; ADAR; Crosslinking reaction; Native-like; RNA.

Figure 1

RNA containing crosslinking base 6-vinylpurine (VP). Expected crosslinking reaction of RNA containing VP with cytosine (C) (top), and with uracil (U) (bottom) on the complementary RNA strand.

Figure 2

HPLC profiles of (A) ORN1 and (B) ORN2.

Figure 3

Crosslinking reaction with the model sequence ORN1–3. Condition screening with (A) ORN1, (B) ORN2 and (C) ORN3. The reaction was performed in MES buffer (50 mM. pH 7.0 or 5.0) containing NaCl (100 mM) at 37 or 50 °C for 24 h. Time course of crosslinking reaction of (D, blue square) ORN1-ORN10(U), pH 7, 50 °C, (D, red circle) ORN1-ORN9(C), pH 5, 37 °C, (E, blue square) ORN1-ORN9(U), pH 5, 50 °C, (E, red circle) ORN1-ORN11(C), pH 5, 50 °C. (F) Gel image of crosslinking formation of ORN2-ORN9(U).

Figure 4

Crosslinking reaction with the ADAR substrate sequence ORN4–6. Condition screening with (A) ORN4, (B) ORN5 and (C) ORN6. The reaction was performed in MES buffer (50 mM. pH 7.0 or 5.0) containing NaCl (100 mM) at 37 or 50 °C for 24 h. Time course of crosslinking reaction of (D) ORN4-ORN13, pH 7, 50 °C, (E) ORN5-ORN13, pH 5, 37 or 50 °C, (F) ORN6-ORN13, pH 5 or 7, 50 °C.

Figure 5

Alkali mediated foot printing of crosslinked ORN2-ORN9(U). 1: ORN9, 2: crosslinked duplex ORN2-ORN9(U), 3: after reaction with ORN9, 4: after reaction with crosslinked duplex ORN2-ORN9(U).

Figure 6

Melting temperature measurement of dsRNA. A: ORN2-ORN9(U) duplex, B: ORN2-ORN11(C) duplex, C: ORN6-ORN13 duplex. Natural duplex and crosslinked duplex are shown as dotted line and solid line, respectively.

Figure 7

CD measurement of dsRNA. A: ORN2-ORN9(U) duplex, B: ORN2-ORN11(C) duplex, C: ORN6-ORN13 duplex. Natural duplex and crosslinked duplex are shown as dotted line and solid line, respectively.

Figure 8

Competitive binding assay result with ADAR2 enzyme. red: native dsRNA; blue: crosslinked dsRNA.

Scheme 1

Synthesis of CPG (4 and 5) and phosphoramidite (7): (a) DMTrCl, pyridine, 88%; (b) tributylvinyltin, PdCl₂(PPh₃)₂, DMF, 77%; (c) NaSMe, H₂O, DMF, 84%; (d) Succinylated CPG support, EDC, DMAP, DIPEA, pyridine, 56%; (e) TBSCl, AgNO₃, THF, 56%; (f) iPr₂NP(Cl)OCH₂CH₂CN, DIPEA, CH₂Cl₂, 92%.

Scheme 2

RNA synthesis and purification. (a) RNA synthesizer, (b) NH₄OH/EtOH (3:1), 30 °C, 12h; (c) TEA-3HF, 65 °C, 2.5 h.

Scheme 3

Synthesis of VP-containing ORN-D. (a) MMPP, H₂O, RT, 30 min, (b) 50% AcOH, 37°C, 3h.