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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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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.
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fig5: 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.

Mentions: The labeling of pRNA for single molecule imaging has been challenging, as single fluorophore labeling at desired nucleotide position is required. Utilizing our unique single labeling strategy coupled with the circular permutation pRNA (cpRNA) technique,48,70 labeling with only one fluorophore at the desired base is ensured. The circular permutation technique generates new 3′/5′ openings at desired base locations along the pRNA sequence.48,70−72 The correct folding of pRNAs after this rearrangement has been extensively tested.38,48,70,73 The single labeling at the new 5′-ends was then achieved by in vitro transcription with a fluorescent AMP.(74) Various RNA molecules (1B, RNA1 in the dimer) were constructed using the circular permutation pRNA approach and labeled with a single Cy3 at various nucleotides (Nt). Each labeled RNA was paired with its partner RNA2 (1B) containing both a Cy5 and a biotin moiety. The biotin label allows for immobilization to a streptavidin coated quartz slide surface for TIRF imaging. The distances between two intermolecular bases within the dimer (1B) were studied by smFRET. Dimers were assembled with high efficiency when pRNA Ab′ was mixed with an equal amount of Ba′, as confirmed by native PAGE gel (4A and B), procapsid binding (4C) and AFM imaging (5).


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)

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

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

fig5: 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.
Mentions: The labeling of pRNA for single molecule imaging has been challenging, as single fluorophore labeling at desired nucleotide position is required. Utilizing our unique single labeling strategy coupled with the circular permutation pRNA (cpRNA) technique,48,70 labeling with only one fluorophore at the desired base is ensured. The circular permutation technique generates new 3′/5′ openings at desired base locations along the pRNA sequence.48,70−72 The correct folding of pRNAs after this rearrangement has been extensively tested.38,48,70,73 The single labeling at the new 5′-ends was then achieved by in vitro transcription with a fluorescent AMP.(74) Various RNA molecules (1B, RNA1 in the dimer) were constructed using the circular permutation pRNA approach and labeled with a single Cy3 at various nucleotides (Nt). Each labeled RNA was paired with its partner RNA2 (1B) containing both a Cy5 and a biotin moiety. The biotin label allows for immobilization to a streptavidin coated quartz slide surface for TIRF imaging. The distances between two intermolecular bases within the dimer (1B) were studied by smFRET. Dimers were assembled with high efficiency when pRNA Ab′ was mixed with an equal amount of Ba′, as confirmed by native PAGE gel (4A and B), procapsid binding (4C) and AFM imaging (5).

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