Interbase FRET in RNA: from A to Z

Abstract

Interbase FRET can reveal highly detailed information about distance, orientation and dynamics in nucleic acids, complementing the existing structure and dynamics techniques. We here report the first RNA base analogue FRET pair, consisting of the donor tCO and the non-emissive acceptor tCnitro. The acceptor ribonucleoside is here synthesised and incorporated into RNA for the first time. This FRET pair accurately reports the average structure of A-form RNA, and its utility for probing RNA structural changes is demonstrated by monitoring the transition from A- to Z-form RNA. Finally, the measured FRET data were compared with theoretical FRET patterns obtained from two previously reported Z-RNA PDB structures, to shed new light on this elusive RNA conformation.

Figure 1.

Structure of emissive FRET donor tC^O and non-emissive FRET acceptor tC_nitro.

Figure 2.

(A) General 3D structures of A- and Z-form RNA. (B) Illustration of the six base step parameters used when building nucleic acid structures. Figure adapted from Lu et al. (20).

Scheme 1.

Synthesis of the tC_nitro phosphoramidite 5. Reagents and conditions: (A) t-Bu₂Si(OTf)₂, DMF, 0°C, 1 h; then imidazole, 0°C, 30 min; then TBS-Cl, RT, 12 h. (B) NH₂NH₂(aq.), EtOH, RT, 18 h. (C) CuI, Cs₂CO₃, DMSO, 60°C, 24 h. (D) PyBOP, DBU, MeCN, 0°C, 1 h; then RT, 4 h. (E) Py·(HF)_x, CH₂Cl₂, 0°C --> RT, 6 h. (F) DMTr-Cl, Py, 0°C, 30 min; then RT, 4 h. (G) CEP-Cl, DIPEA, THF, RT, 20 h.

Figure 3.

(A) RNA sequences used to investigate the interbase FRET characteristics in A-form RNA. (B) RNA sequences used for investigating the transition from A- to Z-form RNA. The DX and AY notation of sequences reflects positions of the donor, tC^O (blue), and non-emissive acceptor, tC_nitro (orange), in the sequence, counting from (A) the 5′-end or (B) the 5′-end of the GC-repeat of the donor-containing sequence. Each sequence combination contained only one donor and a maximum of one acceptor. Abasic sites are denoted with an underscore. For a complete listing of all duplexes used for this study, see Supplementary Tables S1 and S7.

Figure 4.

Spectral overlap between tC^O emission and tC_nitro absorption in A-form RNA. The spectra are normalised at their long-wavelength maxima. Measurements were performed at RT in phosphate buffer, pH 7.4, 123 mM Na⁺.

Figure 5.

FRET efficiency between tC^O and tC_nitro in A-form RNA as a function of base-pair separation. Cyan diamonds mark averaged data from steady-state and lifetime measurements. Black diamonds mark the predicted FRET efficiency for tC^O–tC_nitro inside A-form RNA, with a dashed curve showing the predicted FRET efficiency at non-integer separations. Gray diamonds mark the predicted FRET efficiency for tC^O–tC_nitro inside B-form nucleic acid, with a dashed curve showing the predicted FRET efficiency at non-integer separations. Measurements were performed at RT in phosphate buffer, pH 7.4, 123 mM Na⁺.

Figure 6.

(A) Measured FRET efficiency between tC^O and tC_nitro for the GC-repeat in Z-form (green circles), together with predicted FRET values of previously reported Z-form structures: PDB entries 1T4X (blue triangles, NMR structure of Z-form RNA in 6 M NaClO₄), 2GXB (orange diamonds, crystal structure of Z-form RNA bound to ADAR1) and 1QBJ (red squares, crystal structure of Z-form DNA bound to ADAR1) (31–33). (B) Measured FRET efficiency between tC^O and tC_nitro for the GC-repeat in A-form (black squares) and Z-form (green circles). Arrows indicate the direction of change upon switching from A- to Z-form. Measurements were performed at RT in phosphate buffer, pH 7.4, 123 mM Na⁺ (A-form) or with 8 M NaClO₄ added (Z-form), and are averaged data from steady-state and lifetime measurements.