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.

A prebiotic genetic alphabet as an early Darwinian ancestor for pre-RNA evolution

“…The K + ions in the sensor buffer trigger the G-quadruplex formation and the typical signature of a G-quadruplex was readily observed in the unmodified RNA aptamer (UbSP) as shown in Fig 3C (red trace). The CD spectrum of the UbSP aptamer also revealed a positive band at 270 nm, and smaller negative peaks at 238 nm and 215 nm, indicating the formation of a mixture of the hybrid, parallel/anti-parallel G-quadruplex, and is consistent with the literature 47,48 . Interestingly, a similar CD result was obtained for the modified bSP RNA aptamer as well (BAbSP, Fig 3C , blue trace), in which 10 uridine residues were replaced by a prebiotically pertinent genetic alphabet, barbitudine.…”

A Prebiotic Genetic Nucleotide as an Early Darwinian Ancestor for Pre-RNA Evolution

“…The K + ions in the sensor buffer trigger G-quadruplex formation, and the typical signature of a G-quadruplex was readily observed in the unmodified RNA aptamer (UbSP) as shown in Figure C (red trace). The CD spectrum of the UbSP aptamer also revealed a positive band at 270 nm and smaller negative peaks at 238 and 215 nm, indicating the formation of a mixture of the hybrid, parallel/antiparallel G-quadruplex, and is consistent with what has been reported in the literature. , Interestingly, a similar CD result was obtained for the modified BAbSP RNA aptamer as well (Figure C, blue trace), in which up to 10 uridine residues could have been potentially replaced by the PGN, barbitudine. The overall secondary structure seemed to be in reasonable agreement with the control UbSP aptamer.…”

Structural Basis for Fluorescence Activation by Pepper RNA

“…Mango, , Corn, and Chili have also been developed to fulfill a wide range of needs, from even tighter binding affinities and photo-bleaching resistance to optimization for FRET applications. These fluorogenic aptamers all function by binding and restricting bond rotation of a small-molecule chromophore to inhibit non-radiative pathways of decay and thereby promote fluorescence. − Continued development and improvement of these tools require understanding their structure and biophysical properties.…”