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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.
ACS Nano 10/2010; 4(11):6843-53. · 10.77 Impact Factor
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ABSTRACT: Simultaneous detection of two fluorescent markers is important in determination of distance, relative motion and conformational change of nanoparticles or nanodevices. We constructed an imaging system which combines deep-cooled sensitive EMCCD camera with both the objective- and prism-type TIRF. A laser combiner was introduced to facilitate laser controls for simultaneous dual-channel imaging by deliver lasers with different wavelength synchronically via an optic fiber to the sample. The system produces stable signal with extremely low background fluorescence for single-fluorophore detection. It has been applied to study the structure, stoichiometry, and function of the phi29 DNA packaging motor. Single-molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of pRNA on the motor and elucidated the mechanism of pRNA hexamer assembly. The feasibility of single-molecule FRET with this system was demonstrated. Distance rulers of dual-labeled molecule standards were used to evaluate the system. We have also re-engineered the energy conversion protein, gp16, of phi29 motor for single fluorophore labeling to facilitate the single-molecule studies of motor mechanism. The potential applications of single-molecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with co-localization (SHREC) approaches to the study of the phi29 nanomotor are under investigation.
Proc SPIE 01/2010; 7571:757107-757108.
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ABSTRACT: The bacteriophage phi29 DNA packaging motor contains a protein core with a central channel comprising twelve copies of re-engineered gp10 protein geared by six copies of packaging RNA (pRNA) and a DNA packaging protein gp16 with unknown copies. Incorporation of this nanomotor into a nanodevice would be beneficial for many applications. To this end, extension and modification of the motor components are necessary for the linkage of this motor to other nanomachines. Here the re-engineering of the motor DNA packaging protein gp16 by extending its length and doubling its size using a fusion protein technique is reported. The modified motor integrated with the eGFP-gp16 maintains the ability to convert the chemical energy from adenosine triphosphate (ATP) hydrolysis to mechanical motion and package DNA. The resulting DNA-filled capsid is subsequently converted into an infectious virion. The extended part of the gp16 arm is a fluorescent protein eGFP, which serves as a marker for tracking the motor in single-molecule studies. The activity of the re-engineered motor with eGFP-gp16 is also observed directly with a bright-field microscope via its ability to transport a 2-microm-sized cargo bound to the DNA.
Small 10/2009; 5(21):2453-9. · 8.35 Impact Factor
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ABSTRACT: A customized laser combiner was designed and constructed for dual channel single molecule imaging. The feasibility of a combiner-incorporated imaging system was demonstrated in studies of single molecule FRET. Distance rulers made of dual-labeled dsDNA were used to evaluate the system by determining the distance between one FRET pair. The results showed that the system is sensitive enough to distinguish between distances differing by two base pair and the distances calculated from FRET efficiencies are close to those documented in the literature. The single molecule FRET with the dual-color imaging system was also applied to reconstructed phi29 motor pRNA monomers. Finally, techniques for dual laser alignment and tuning of laser power for dual-color excitation are discussed.
Biomedical Microdevices 10/2009; 12(1):97-106. · 3.03 Impact Factor
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ABSTRACT: A dual-channel imaging system with single fluorophore sensitivity was assembled in this lab. Inclusion of an integrated laser combiner was introduced to facilitate simultaneous dual-channel imaging. The imaging system has been applied to study the structure, stoichiometry, distance and function of the phi29 DNA packaging motor. Approaches including single molecule photobleaching, single molecule FRET and binomial distribution quantification were carried out to clarify the stoichiometry and distance of pRNA on the biologically active packaging motor. The results were statistically analyzed to access the copy number of pRNA, and the distance constraint was used to verify the 3D structure of the computer model of phi29 DNA packaging motor.
IEEE/NIH Life Science Systems and Applications Workshop. IEEE/NIH Life Science Systems and Applications Workshop. 04/2009; 2009:124-127.
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ABSTRACT: We report a simple and robust magnetomechanical system for direct visual observation of the DNA packaging behavior of the bacteriophage phi29 in real time. The system comprises a micron-sized magnetic bead attached to the free end of the viral DNA, a magnet and a bright-field microscope. We show that the phi29 DNA packaging activity can be observed and dynamically analyzed at the single molecular level in bright field with a relatively simple system. With this system we also visually demonstrate the phi29 motor transporting a cargo 10 000 times the viral size.
Applied Physics Letters 11/2008; 93(15):153902. · 3.84 Impact Factor
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ABSTRACT: Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.
Nucleic Acids Research 11/2008; 36(20):6620-32. · 8.03 Impact Factor
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ABSTRACT: Similar to the assembly of other dsDNA viruses, bacterial virus phi29 uses a motor to translocate its DNA into a procapsid, with the aid of protein gp16 that binds to pRNA 5'/3' helical region. To investigate the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was evaluated as a function of DNA structure (ss- or ds-stranded) or chemistry (purine or pyrimidine). The k(cat) and K(m) in the absence of DNA was 0.016 s(-1) and 351.0 microM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of DNA or pRNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine)>dsDNA>poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involved in the contact with gp16 during DNA packaging.
Virology 09/2008; 380(1):69-74. · 3.35 Impact Factor
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ABSTRACT: Limited by the spatial resolution of optical microscopy, direct detection or counting of single components in biological complexes or nanoparticles is challenging, especially for RNA, which is conformationally versatile and structurally flexible. We report here the assembly of a customized single-molecule dual-viewing total internal reflection fluorescence imaging system for direct counting of RNA building blocks. The RNA molecules were labeled with a single fluorophore by in vitro transcription in the presence of a fluorescent AMP. Precise calculation of identical or mixed pRNA building blocks of one, two, three, or six copies within the bacteriophage phi29 DNA packaging motor or other complexes was demonstrated by applying a photobleaching assay and evaluated by binomial distribution. The dual-viewing system for excitation and recording at different wavelengths simultaneously will enable the differentiation of different complexes with different labels or relative motion of each labeled component in motion machines.
RNA 11/2007; 13(10):1793-802. · 5.09 Impact Factor
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ABSTRACT: Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA-packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20-nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual-color detection of the stalled intermediates of motors containing Cy3-pRNA and Cy5-DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat-denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.
The EMBO Journal 02/2007; 26(2):527-37. · 9.20 Impact Factor
