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Dual-channel single-molecule fluorescence resonance energy transfer to establish distance parameters for RNA nanoparticles.

Shu D, Zhang H, Petrenko R, Meller J, Guo P - ACS Nano (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiomedical Center, College of Engineering and Applied Science/College of Medicine, University of Cincinnati, Cincinnati, Ohio 45221, United States.

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.

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Related in: MedlinePlus

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.
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fig2: 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.

Mentions: The distance range between the donor and the acceptor to achieve measurable energy transfer is approximately 1−7.5 nm for a Cy3/Cy5 pair.(24) The labeling of RNA with fluorescent dyes generates tethered arms whose length is an important factor in distance determination by FRET. However, it is difficult to obtain a solid value of arm size for each fluorophore due to other factors such as orientation, folding and flexibility in molecular arrangement. Empirical determination is one feasible approach to estimate the length for both the donor and acceptor arms. Hence, we used five standard RNA/DNA hybrids with known distances between the donor and acceptor (12bp, 14bp, 16bp, 18bp, and 20bp) (2A) to determine the empirical arm size of the fluorophore and to evaluate the feasibility of using our dual-viewing single molecule imaging system to study pRNA structure. The RNA/DNA hybrids were chosen here as the labeled pRNA dimers studied in this report were partially constructed through RNA/DNA annealing.


Dual-channel single-molecule fluorescence resonance energy transfer to establish distance parameters for RNA nanoparticles.

Shu D, Zhang H, Petrenko R, Meller J, Guo P - ACS Nano (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2990273&req=5

fig2: 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.
Mentions: The distance range between the donor and the acceptor to achieve measurable energy transfer is approximately 1−7.5 nm for a Cy3/Cy5 pair.(24) The labeling of RNA with fluorescent dyes generates tethered arms whose length is an important factor in distance determination by FRET. However, it is difficult to obtain a solid value of arm size for each fluorophore due to other factors such as orientation, folding and flexibility in molecular arrangement. Empirical determination is one feasible approach to estimate the length for both the donor and acceptor arms. Hence, we used five standard RNA/DNA hybrids with known distances between the donor and acceptor (12bp, 14bp, 16bp, 18bp, and 20bp) (2A) to determine the empirical arm size of the fluorophore and to evaluate the feasibility of using our dual-viewing single molecule imaging system to study pRNA structure. The RNA/DNA hybrids were chosen here as the labeled pRNA dimers studied in this report were partially constructed through RNA/DNA annealing.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiomedical Center, College of Engineering and Applied Science/College of Medicine, University of Cincinnati, Cincinnati, Ohio 45221, United States.

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.

Show MeSH
Related in: MedlinePlus