Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

Abstract

The increasing interest in RNA nanotechnology and the demonstrated feasibility of using RNA nanoparticles as therapeutics have prompted the need for imaging systems with nanometer-scale resolution for RNA studies. Phi29 dimeric pRNAs can serve as building blocks in assembly into the hexameric ring of the nanomotors, as modules of RNA nanoparciles, and as vehicles for specific delivery of therapeutics to cancers or viral infected cells. The understanding of the 3D structure of this novel RNA dimeric particle is fundamentally and practically important. Although a 3D model of pRNA dimer has been proposed based on biochemical analysis, no distance measurements or X-ray diffraction data have been reported. Here we evaluated the application of our customized single-molecule dual-viewing system for distance measurement within pRNA dimers using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Ten pRNA monomers labeled with single donor or acceptor fluorophores at various locations were constructed and eight dimers were assembled. smFRET signals were detected for six dimers. The tethered arm sizes of the fluorophores were estimated empirically from dual-labeled RNA/DNA standards. The distances between donor and acceptor were calculated and used as distance parameters to assess and refine the previously reported 3D model of the pRNA dimer. Distances between nucleotides in pRNA dimers were found to be different from those of the dimers bound to procapsid, suggesting a conformational change of the pRNA dimer upon binding to the procapsid.

Diverse self-association properties within a family of phage packaging RNAs

“…The crystal structure of the isolated phi29 3wj core showed similar helical arrangements, with some difference in the angle between P A and P C (Zhang et al 2013). Interestingly, when the phi29 pRNA was studied using solution methods such as single-molecule FRET and electron paramagnetic resonance, the resultant models show a coaxial stacking of P C and P D , with P A emerging at an angle, a conformation also suggested by computer modeling (Hoeprich and Guo 2002;Shu et al 2010;Zhang et al 2012). In addition, it has been shown that the UUU triplet at nucleotides 50-52 in the phi29 pRNA 3wj is important for helical conformation and phage DNA packaging, leading to the proposal that inherent flexibility in the junction is important for function (Zhao et al 2012).…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…The sample was immobilized at the biotin-BSA-coated surface of the sample chamber via biotin/streptavidin interaction according to methods previously described (Shu et al 2010). Concentration of the samples was carefully picked to ensure the observation of individual fluorescent spots while keeping sufficient number of these spots in each field of view.…”

“…Concentration of the samples was carefully picked to ensure the observation of individual fluorescent spots while keeping sufficient number of these spots in each field of view. Prism-type total internal reflection fluorescence (TIRF) imaging was performed according to methods previously described (Shu et al 2010). A laser beam of wavelength 532 nm was used to excite the FRET pair, and dual-color fluorescence images were taken.…”

Intracellular pH Measurements Using Perfluorocarbon Nanoemulsions

“…For this pair, energy transfer can be observed when the donor and acceptor are within 8 nm. 29 Since CypHer5 and Cy5 are spectrally similar when CypHer5 is protonated, there is potential for FRET due to the Cy3-CypHer5 pairing. Efficient energy transfer could potentially complicate pH calibrations and estimation of the localized cellular pH of internalized nanoemulsions.…”