title: Visualising single molecules of HIV-1 and miRNA nucleic acids.
abstract: 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.
authors.affiliation: Centre for Virology, Burnet Institute, Melbourne, Australia.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Jones
authors.forename: Adam
authors.initials: A
authors.lastname: Karpala
authors.forename: Bevan
authors.initials: B
authors.lastname: Hirst
authors.forename: Kristie
authors.initials: K
authors.lastname: Jenkins
authors.forename: Mark
authors.initials: M
authors.lastname: Tizard
authors.forename: Cândida F
authors.initials: CF
authors.lastname: Pereira
authors.forename: Andrew
authors.initials: A
authors.lastname: Leis
authors.forename: Paul
authors.initials: P
authors.lastname: Monaghan
authors.forename: Alex
authors.initials: A
authors.lastname: Hyatt
authors.forename: Johnson
authors.initials: J
authors.lastname: Mak
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin / Q000276:immunology; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics / Q000648:ultrastructure*; D004279:DNA, Viral / Q000235:genetics / Q000648:ultrastructure*; D018628:Gene Dosage / Q000235:genetics; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D019169:Jurkat Cells; D035683:MicroRNAs / Q000235:genetics / Q000648:ultrastructure*; D008853:Microscopy / Q000379:methods; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000235:genetics / Q000648:ultrastructure*
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004279:DNA, Viral; D035683:MicroRNAs; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D001710:Biotin