Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy.
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.
Noestheden
Matthew
M
Steacie Institute for Molecular Sciences, The National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada.
Hu
Qingyan
Q
Tonary
Angela M
AM
Tay
Li-Lin
LL
Pezacki
John Paul
JP
D055592:Biophysical Phenomena; D001703:Biophysics; D045744:Cell Line, Tumor; D003287:Contrast Media; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008853:Microscopy; D015335:Molecular Probes / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry*; D012093:Replicon / Q000235:genetics; D013059:Spectrum Analysis, Raman; D013194:Staining and Labeling / Q000379:methods*; D014529:Uridine / Q000235:genetics; D014732:Vibration
D003287:Contrast Media; D015335:Molecular Probes; D009570:Nitriles; D012367:RNA, Viral; C014356:benzonitrile; D014529:Uridine