Synthesis of 6-Se-guanosine RNAs for structural study

Abstract

6-Se-guanosine phosphoramidite and RNAs have been synthesized by selenium substitution of the 6-oxygen atom, and it is revealed that the Se-derivatization is relatively stable and that bulge and wobble structures can better accommodate a large Se atom than a duplex. This Se-modification is useful in the structural study of RNAs and their protein complexes.

Figure 1

HPLC analysis and thermostability study. The purified RNA samples [^SeG-RNA: r(5′-G-^SeG-UAUUGCGGUACC-3′); 3 μM each] were heated at 60 °C in 100 mM sodium phosphate buffer (pH 7.6) for 1, 2 and 3 hours. (A) RP-HPLC analysis at 360 nm, no heat; (B) 1-hr heating (360 nm); (C) 2-hr heating (360 nm); (D) 3-hr heating (360 nm); (E) 3-hr heating (260 nm) in blue line; 3-hr heating (360 nm) in red line. HPLC conditions: Welchrom XB-C18 column (4.6×250 mm); flow rate: 1 mL/min; column temperature: 25 °C; gradient: 5 to 35% B in 10 min; Buffer A: 10 mM TEAAc (pH 7.4); Buffer B: 10 mM TEAAc (pH 7.4) in 60% acetonitrile dissolved in water (6:4 in v/v).

Figure 2

The RNA secondary structures and normalized UV-melting curves of RNA 1-6 in Table 2. (A) The secondary structures of RNAs: 1 (native in blue), 2 (with black ^SeG), and 3 (with red ^SeG). (B) The secondary structures of RNAs: 4 (native in blue), 5 (with black ^SeG), and 6 (with red ^SeG). The underline sequences are U/G wobble pairs. (C) the blue curve: melting curve of the native RNA containing two adenosine bulges (1; T_m= 39.0 °C); the black curve: melting curve of 2 (T_m= 38.5 °C); the red curve: melting curve of 3 (T_m= 36.5 °C). The melting temperatures of the duplexes (2.5 μM) were measured in 10 mM phosphate buffer (pH 6.5) containing 50 mM NaCl. The absorbance of the samples was monitored at 260 nm, and the duplexes were headed from 5 to 70 °C with a rate of 1 °C/min.

Figure 3

The structure comparison and crystal picture of the RNA/DNA/RNase H complexes. (A) The Se-transient native structure (resolution: 1.60 Å; PDB: 3ULD) is in cyan (protein) and red (RNA/DNA duplex), while the direct native structure (resolution: 2.70 Å; PDB: 2G8U) is in grey (protein) and green (RNA/DNA duplex). The sequences of the RNA (5′-UCGACA-3′) and DNA (5′-ATGTCG-3′) are the same in these two structures. (B) Picture of the crystal (0.2×0.2×0.2 mm; slightly yellow) of the transient Se-RNA/DNA/RNase H complex.

Scheme 1

Synthesis of SeG-RNAs (5)