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Published online before print August 13, 2007, 10.1261/rna.587607
RNA (2007), 13:1793-1802. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.
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METHOD

Instrumentation and metrology for single RNA counting in biological complexes or nanoparticles by a single-molecule dual-view system

Hui Zhang1,2, Dan Shu1,2, Faqing Huang3, and Peixuan Guo1,2

1 Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA
2 Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA
3 Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi 39406, USA

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.

Keywords: single or dual labeling; bacteriophage phi29 motor pRNA; viral DNA packaging motor; photobleaching

Received March 12, 2007 ; accepted July 10, 2007.


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