Visualising single molecules of HIV-1 and miRNA nucleic acids
AuthorJones, KL; Karpala, A; Hirst, B; Jenkins, K; Tizard, M; Pereira, CF; Leis, A; Monaghan, P; Hyatt, A; Mak, J
Source TitleBMC Cell Biology
University of Melbourne Author/sLeis, Andrew
Document TypeJournal Article
CitationsJones, K. L., Karpala, A., Hirst, B., Jenkins, K., Tizard, M., Pereira, C. F., Leis, A., Monaghan, P., Hyatt, A. & Mak, J. (2013). Visualising single molecules of HIV-1 and miRNA nucleic acids. BMC CELL BIOLOGY, 14 (1), https://doi.org/10.1186/1471-2121-14-21.
Access StatusOpen Access
BACKGROUND: The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19-26 nucleotides in length. RESULTS: We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1-2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. CONCLUSIONS: Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1-2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.
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