Photophysics of DFHBI bound to RNA aptamer Baby Spinach

Abstract

The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system.

Figure 1

Absorption (A) and fluorescence (B) spectra (λ_exc 460 nm) of free DFHBI (2 µM) in the absence of bSP (black) and its mixture with increasing bSP concentrations, all measured in a flow-cell at low intensity. (C) Fluorescence excitation spectra (λ_probe 501 nm) of free DFHBI and in the presence of increasing bSP concentrations. (D) Normalized calculated absorption spectra for increasing bSP concentrations as derived from K_d = 4.4 µM in comparison with the fluorescence excitation spectrum (red, dotted).

Figure 2

(A) Comparison of experimental and calculated fluorescence intensity. The continuous line represents the best fit of the experimental data to the multiple-site model. (B) The normalized calculated absorption spectrum of the DFHBI/bSP complex derived with K_d = 7.9 µM in comparison to the observed fluorescence excitation spectrum (Fig. 1D).

Figure 3

Absorption spectra of the system DFHBI/bSP at saturating conditions 2 µM DFHBI/20 µM bSP. (A) (a) Measured absorption spectrum of the mixture Fig. 1A (red), (b) calculated absorption spectrum of the fluorescing complex (D•RNA)_FL (brown), and (c) calculated absorption spectrum of free dye and dark complexes (D•RNA)_DARK (blue). (B) Absorption spectrum of the free dye (black) and absorption spectrum of the mixture of free dye and non-fluorescent complex from (A) (blue). Note that the absorption spectrum of the mixture is narrowed and slightly red shifted from 416 to 419 nm.

Figure 4

Fluorescence decay probed at 500 nm of DFHBI (2 μM) in absence (black) and presence (red) of bSP (20 μM) under flow cell conditions. (A) Time Correlated Single Photon Counting (TCSPC) measurements of the free dye (black) in solution excited at 416 nm and of the complex excited at 470 nm (red), excitation power 10 μW.The fluorescence decay of the free dye follows the instrument function IRF and is resolved in Fig. 4B. (B) Fluorescence up-conversion measurements of the free DFHBI (black) and the DFHBI/bSP mixture (red), excitation at 400 nm (25 mW).