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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

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.
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fig4: 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.

Mentions: The feasibility of applying FRET for RNA structure studies is a case by case issue. RNAs will refold partially or completely when a new chemical group, dye or nucleotide is introduced into the RNA sequence. It is not feasible to simply introduce a pair of dyes to the RNA and measure the FRET to derive the distance. However in this work, after each dye introduced into each pRNA molecule, we assess their structures and folding by at least one of the following approaches: 1. dimer formation;36,61 2. procapsid binding;31,62,63 3. DNA packaging activity;64,65 4. in vitro phi29 virion assembly activity (4).66,67 The mutated or truncated RNA with incomplete pRNA sequence would be inactive in DNA packaging or in virion assembly. Thus, using biological activity to assess their folding is not possible. In such cases, we only tested their binding to procapsid using [3H] RNA (4C). If the pRNA failed in any one of the functions, we regarded the pRNA as structurally refolded and did not consider the data comparable to the wild type pRNA.


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)

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

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

fig4: 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.
Mentions: The feasibility of applying FRET for RNA structure studies is a case by case issue. RNAs will refold partially or completely when a new chemical group, dye or nucleotide is introduced into the RNA sequence. It is not feasible to simply introduce a pair of dyes to the RNA and measure the FRET to derive the distance. However in this work, after each dye introduced into each pRNA molecule, we assess their structures and folding by at least one of the following approaches: 1. dimer formation;36,61 2. procapsid binding;31,62,63 3. DNA packaging activity;64,65 4. in vitro phi29 virion assembly activity (4).66,67 The mutated or truncated RNA with incomplete pRNA sequence would be inactive in DNA packaging or in virion assembly. Thus, using biological activity to assess their folding is not possible. In such cases, we only tested their binding to procapsid using [3H] RNA (4C). If the pRNA failed in any one of the functions, we regarded the pRNA as structurally refolded and did not consider the data comparable to the wild type pRNA.

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