Förster resonance energy transfer within the neomycin aptamer

• Published in: Physical chemistry chemical physics : PCCP 2024-02, Vol.26 (8), p.7157-7165
• Main Authors: Hurter, Florian, Halbritter, Anna-Lena J, Ahmad, Iram M, Braun, Markus, Sigurdsson, Snorri Th, Wachtveitl, Josef
• Format: Article
• Language: English
• Subjects: Absorption; Biopolymers; Dyes; Energy transfer; Fluorescence; Labels; Ligands; NMR; Nuclear magnetic resonance; Resonance; Spatial resolution; Time correlation functions
• ISSN: 1463-9076; 1463-9084
• DOI: 10.1039/d3cp05728c

Förster resonance energy transfer (FRET) measurements between two dyes is a powerful method to interrogate both structure and dynamics of biopolymers. The intensity of a fluorescence signal in a FRET measurement is dependent on both the distance and the relative orientation of the dyes. The latter can at the same time both complicate the analysis and give more detailed information. Here we present a detailed spectroscopic study of the energy transfer between the rigid FRET labels Ç m f (donor) and tC nitro (quencher/acceptor) within the neomycin aptamer N1. The energy transfer originates from multiple emitting states of the donor and occurs on a low picosecond to nanosecond time-scale. To fully characterize the energy transfer, ultrafast transient absorption measurements were performed in conjunction with static fluorescence and time-correlated single photon counting (TCSPC) measurements, showing a clear distance dependence of both signal intensity and lifetime. Using a known NMR structure of the ligand-bound neomycin aptamer, the distance between the two labels was used to estimate κ 2 and, therefore, make qualitative statements about the change in orientation after ligand binding with unprecedented temporal and spatial resolution. The advantages and potential applications of absorption-based methods using rigid labels for the characterization of FRET processes are discussed. A methodological analysis of FRET data from a new pair of modified nucleobases, Ç m f and tC nitro , for energy transfer in RNA aptamers. The limitations and strengths of different methods are highlighted, depending on the energy transfer time scale.