Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions.
Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription-polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.
Faridani
Omid R
OR
Department of Cell and Molecular Biology, Karolinska Institutet, Berzelius väg 35, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, S-171 77, Stockholm, Sweden. omid.faridani@ki.se
McInerney
Gerald M
GM
Gradin
Katarina
K
Good
Liam
L
D001427:Bacterial Toxins / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005821:Genetic Techniques; D016325:HIV Long Terminal Repeat; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C087025:Hok protein, E coli; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D011088:DNA Ligases