ABSTRACT
Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.
Subject(s)
In Situ Hybridization/methods , Transglutaminases/metabolism , Animals , Mice , Protamines/genetics , Pyrococcus furiosus/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Uromodulin/geneticsABSTRACT
A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.
Subject(s)
DNA/metabolism , Pyrococcus furiosus/enzymology , Transglutaminases/metabolism , Alkaline Phosphatase/metabolism , Catalysis , DNA/analysis , DNA/chemistry , Deoxyuracil Nucleotides/chemistry , Dipeptides/metabolism , Electrophoresis, Agar Gel , Glutamine/chemistry , Lysine/chemistry , Peptides/chemical synthesis , Peptides/chemistryABSTRACT
We determined the subtype of HIV-1 in 89 infected individuals attending three reference hospitals located in the Tokyo metropolitan area of Japan. Subtyping was performed with subtype-specific polymerase chain re-action (PCR) distinguishing subtype A, B, C, and CRF01_AE and/or phylogenetic analysis of HIV-1 env C2V3C3 sequences. Subtype-specific PCR provided unequivocal results in 97% of samples. Sixty-five subjects were infected with subtype B, 16 with CRFO1_AE, 4 with subtype A, 1 with CRF02_AG, and 3 with subtype C. Among 31 Japanese individuals infected through heterosexual contact, 13 were infected with subtype Band 12 with CRFO1_AE. All of the 41 Japanese men infected through homosexual contact harbored subtype B. These results indicate that subtype B is exclusively predominant in a homosexually transmitted group, whereas subtype B and CRFO1_AE are evenly predominant in a heterosexually transmitted group.
