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.

Figure 1

Illustration of (A) sequence and (B) structure of the pRNA dimer. The same letters in upper and lower case indicate complementary sequences for the pRNA loop/loop interaction, while different letters indicate noncomplementary loops. For example, pRNA Ab′ represents pRNA where right loop A (5′G45G46A47C48) is complementary to left loop a′ (3′C85C84U83G82) of pRNA Ba′; and left loop b′ (3′U85G84C83G82) is complementary to right loop B (5′A45C46G47C48). The RNA1 and RNA2 were assembled into dimer via the interlocking loops. Numbers in green represent the six donor positions in subunit Ab′ and numbers in red represent the two acceptor positions in subunit Ba′ in two different dimers (see 2).

Figure 2

Standard distance determination of dual-labeled RNA/DNA hybrids. (A) Design of the dual-labeled RNA/DNA hybrids with different lengths of 12, 14, 16, 18, and 20 bp between Cy3 and Cy5. (B) Typical time trajectory of fluorescence intensity for a FRET event. (C) Histograms summarizing FRET efficiencies of RNA/DNA hybrids 12, 14, 16, 18, and 20 bp (a−e), respectively. (D) Histograms summarizing calculated distances of RNA/DNA hybrids 12, 14, 16, 18, and 20 bp (a−e), respectively, from FRET efficiency.

Figure 3

Empirical determination of the arm size of two fluorophores serving as donor and acceptor. (A) Relationship of FRET efficiencies and numbers of basepairs between the FRET pair. The error bars are the standard deviations of the measurements. (B) Comparison of empirical and theoretical distances to derive the arm size of the donor plus the acceptor fluorophores. The distances derived from FRET efficiency (black) were correlated with the theoretical distances (red) calculated from the value of 0.275 nm per rise of one basepair. The blue dotted lines indicate the empirical arm sizes for donor plus acceptor obtained from the differences.

Figure 4

Verification of pRNA structure and function in (A−B) dimer formation and (C) procapsid binding after modification. (A) Native gel electrophoresis of modified pRNAs, compared with unmodified pRNAs. (B) Fluorescent gel images of modified pRNAs, compared with unmodified pRNAs. (C) Comparison of procapsid binding activities by [3H] counting for the modified pRNAs with unmodified pRNAs. The number of Nt indicates the position of the nucleotides for fluorescent labels.

Figure 5

Comparison of AFM images with the 3D computer model of the pRNA dimer. (A) AFM images showing pRNA dimers. Scale bar: 100 nm. (B) Zoomed AFM images of individual pRNA dimers, compared with 3D modeling images of the pRNA dimer.

Figure 6

Distance determination of pRNA dimers corresponding to pRNA dimer a−f shown in 1. (A) Distribution of FRET efficiencies. (B) Summary of calculated distances of different pRNA dimers.

Figure 7

Refined 3D model of pRNA dimer. (A) Comparison of the original (red) and the refined (blue) models. (B) Refined structure showing positions of nucleotides used for labeling. (C) Elucidation of the location of two nucleotides that were labeled with the donor and the acceptor, respectively, in individual dimer (a−h), with one pRNA subunit in light green ribbon and the other subunit in light red. The bases used for distance measurements are in dark red spacefill format in RNA1 and blue in RNA2. Numbers represent the sequences of each nucleotide. The distances measured from FRET efficiency are indicated.

Figure 8

Comparison of the pRNA head loop structure in our 3D model (yellow) with a newly published pRNA head loop structure by NMR (blue). The red circle indicates the flexible loop region of the head loop structure. See 1A for the complete sequence of pRNA.

Figure 9

Comparison of ensemble FRET of purified dimer composed of pRNA Ab′ (Cy3 at Nt21) and Ba′ (Cy5 at Nt1) with that of the dimer bound to procapsid by their fluorescence emission spectra at 530 nm excitation.