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Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Sequence Specific Single Molecule Tracking of MicroRNA and Messenger RNA Using Quantum Dots

Despite advances in RNA imaging, it remains a challenge to track single native RNA molecules in living cells. The major obstacle is that traditional organic dyes used for RNA imaging fluoresce weakly and are subject to quick photobleaching. Quantum dots are fluorescent semiconductor nanocrystals with much higher brightness and much greater photostability. Here we use quantum dots to image single messenger RNA and microRNA. We have developed an optical imaging technique by which only target RNA-bound quantum dots give positive signals. The quantum dot probes recognize target RNA molecules in a sequence specific manner. Such that microRNA and mRNA are tracked at single molecule level in real time in homogeneous solution. The capability of tracking single RNA molecules allows for counting very low copy numbers of RNA (one or several copies) with very high accuracy and for quantification of RNA with ultrahigh sensitivity. Moreover, the method enables the tracking of single molecule reactions. As one example, we followed the site-specific cleavage of single mRNA by RNase H at DNA oligonucleotide binding sites. Multiple color imaging enables determination of the order of multiple cleavage events at different sites on a single mRNA molecule. Our results demonstrate the potential of the technique in tracking and quantification of RNA, both in vitro and in vivo.

J. Jeffery Li1
Kenneth S. Kosik2
Kaiqin Lao3
X. Sunney Xie1,*

1Department of Chemistry and Chemical Biology
Harvard University
Cambridge, MA 02138, USA
2Department of Neurology
Brigham and Women?s Hospital
Harvard Medical School
Boston, MA 02115, USA
3Applied Biosystems
Foster City
CA 94404, USA

Phone: 617-496-9925
Fax: 617-496-8709
Email: xie@chemistry.harvard.edu