Abstract
Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.
Highlights
As cornerstones of the central dogma and fundamental players in gene regulation, nucleic acids and their structures, dynamics, conformational changes and interactions with other biomolecules is key to the understanding of living organisms
This rebuilding routine is demonstrated by Protein Data Bank (PDB) entry 1TGH, the complex formed between the TATA-binding protein (TBP) and its DNA target (Figure 3c and Supplementary Data S1) [36]
We have developed a general methodological platform for simulating Forster resonance energy transfer (FRET) in nucleic acids and demonstrated its particular power in modelling probes possessing limited degree of diffusional and rotational freedom
Summary
As cornerstones of the central dogma and fundamental players in gene regulation, nucleic acids and their structures, dynamics, conformational changes and interactions with other biomolecules is key to the understanding of living organisms. High-resolution structural insight into nucleic acids is accomplished using nuclear magnetic resonance (NMR) spectroscopy [1] or X-ray crystallography [2], often being complemented by lowerresolution techniques, such as Forster resonance energy transfer (FRET) [3,4]. Because of limitations in available dyes [6], by far the most FRET experiments use external fluorophores being tethered to the nucleic acid through a linker, introducing dye diffusion and reorientation hampering the interpretation of quantitative experiments [7,8,9]. Recent advances have progressed the modelling of linker flexibility in quantitative FRET measurements [10,11,12,13], external labelling will always be accompanied by an inherent limitation in the information obtainable from the technique
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