Photophysics of DFHBI bound to RNA aptamer Baby Spinach

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. Nanyang Technological University National Research Foundation (NRF) Published version This research was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.02-2018.11 (to Nguyen Thuan Dao), Nanyang Technological University grants to M.-E. Michel-Beyerle and to Anh Tuan Phan. Alexander Voityuk is grateful for financial support from the MINECO, Spanish Ministerio de Economía y Competitividad. This work was supported in part by the Singapore National Research Foundation under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.