FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

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.

Implementation of Probabilistic Algorithms by Multi‐chromophoric Molecular Networks with Application to Multiple Travelling Pathways

“…In our model, we consider dyes which are covalently conjugated to nucleotides via C3 or C6 flexible linkers, for which isotropic averaging is appropriate in most cases and . However, other fluorescent probes have been designed for allowing the control of both their position and mutual orientation, such as the tC dye family, intercalating dyes from the YO (oxazole yellow) and TO (thiazole orange) families and intercalated and covalently‐conjugated porphyrins . The control over relative orientations allows enhancing or suppressing the rate of specific energy transfer pathways by changing the value of the orientation factor and it is another means to tune the rate parameters of the random walk dynamics.…”

Computational modeling of RNA 3D structures, with the aid of experimental restraints

“…Several such fluorophores are even commercially available in the form or nucleotide precursors for phosphoramidite oligonucleotide synthesis. Theoretically, for a sufficiently large number of RNA variants, labeled at various helical regions of the RNA structure, the mutual positions and orientations of helices can be determined based on FRET data alone 66 . However, the disadvantage of the currently available fluorescent nucleotide base analogs is their low quantum yield, which makes single molecule measurements relatively impractical.…”

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing

“…[19] The limited brightness of existing fluorescent nucleoside analogues explainsw hy they are not widely used in single-molecule fluorescencem easurements, and the only existing FRET pairs between nucleobasea nalogues are non-emissive. [20][21][22] Ongoing efforts by an umber of groups are focused on developing nucleoside analogues with useful fluorescence changes in response to base stacking, base pairing, changes in nucleic acid secondary and tertiary structure, as well as environmental changes associatedw ith protein binding or changes in nucleic acids olvation. [1,5,[23][24][25][26][27][28][29][30][31] But probablyt he greatesti mpediment to the development of new generationso ff luorescent nucleoside analogues with radically improved properties is that robust methods for predictingp hotophysics based on structure are largely unavailable, especially when one is targeting the complex, anisotropic environment of the base stack.…”

“…[42] These compounds have been used in biophysical studies of enzymem echanisms and Wilhelmsson has more recently developed aF RET pair with tC O as ad onor and nitro-tCa sanon-emissive quencher. [20,21] In seekingt oa dd new properties to the fluorescent nucleoside toolkit,o ur lab synthesized and studied an extended family of tC compounds that collectively hint at structure-photophysics relationships. [19,36] Thatw ork identified 8-Cl-tC O as the member of the tC family with the brightest fluorescence and 8-DEA-tC as the least bright nucleoside, and those results hinted at trends relatinga nalogues'e lectronics to photophysical properties such as Stokes shift.…”