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Article

  • The EMBO Journal (2007) 26, 527 - 537
  • doi:10.1038/sj.emboj.7601506

Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system

Dan Shu1,a, Hui Zhang1,a, Jiashun Jin2 and Peixuan Guo1

  1. Department of Comparative Pathobiology and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA
  2. Department of Statistics, Purdue University, West Lafayette, IN, USA

Correspondence to:

Peixuan Guo, Purdue Cancer Center, Hansen Life Science Research Building, Purdue University, West Lafayette, IN 47907, USA. Tel.: +1 765 494 7561; Fax: +1 765 496 1795; E-mail: guop@purdue.edu

aThese authors contributed equally and share the first authorship

Received 18 August 2006; Accepted 23 November 2006

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.

  • Keywords:

    • biomotor,
    • bionanotechnology,
    • nanobiotechnology,
    • SMDV-TIRF,
    • RNA nanotechnology