Issue 15, 2007
From the journal:

Organic & Biomolecular Chemistry

Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy†

Matthew Noestheden,ab   Qingyan Hu,a   Angela M. Tonary,a   Li-Lin Tayc  and  John Paul Pezacki*ab  

* Corresponding authors

a Steacie Institute for Molecular Sciences, The National Research Council of Canada, 100 Sussex Dr., Ottawa, Ontario, Canada
E-mail: John.Pezacki@nrc.ca
Fax: +1 (613) 952-0068

b Department of Chemistry, University of Ottawa, 10 Marie-Curie, Ottawa, Ontario, Canada

c Institute for Microstructural Sciences, National Research Council of Canada, 207-1200 Montreal Rd., Ottawa, Ontario, Canada

Abstract

Raman and coherent anti-Stokes Raman scattering (CARS) microscopies have the potential to aid in detailed longitudinal studies of RNA localization. Here, we evaluate the use of carbon–deuterium and benzonitrile functional group labels as contrast agents for vibrational imaging of hepatitis C virus (HCV) replicon RNA. Dynamic light scattering and atomic force microscopy were used to evaluate the structural consequences of altering HCV subgenomic replicon RNA. Modification with benzonitrile labels caused the replicon RNA tertiary structure to partially unfold. Conversely, deuterium-modified replicon RNA was structurally similar to unmodified replicon RNA. Furthermore, the deuterated replicon RNA provided promising vibrational contrast in Raman imaging experiments. The functional effect of modifying subgenomic HCV replicon RNA was evaluated using the luciferase gene as a genetic reporter of translation. Benzonitrile labeling of the replicon RNA prevented translation in cell-based luciferase assays, while the deuterated replicon RNA retained both translation and replication competency. Thus, while the scattering cross-section for benzonitrile labels was higher, only carbon–deuterium labels proved to be non-perturbative to the function of HCV replicon RNA.

Graphical abstract: Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy

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Article information

DOI
https://doi.org/10.1039/B704812B
Article type
Paper
Submitted
30 Mar 2007
Accepted
04 Jun 2007
First published
21 Jun 2007

Org. Biomol. Chem., 2007,5, 2380-2389

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Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy

M. Noestheden, Q. Hu, A. M. Tonary, L. Tay and J. P. Pezacki, Org. Biomol. Chem., 2007, 5, 2380 DOI: 10.1039/B704812B

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