Naoaki Okamoto

Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.

Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N‐terminal domain of PriA possesses a binding pocket for the 3′‐terminal nucleotide residue of DNA. The interaction with the deoxyribose 3′‐OH is essential for the 3′‐terminal recognition. In contrast, the direct interaction with 3′‐end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3′ end. Thus, the N‐terminal domain of PriA recognizes the 3′‐end base in a base‐non‐selective manner, in addition to the deoxyribose and 5′‐side phosphodiester group, of the 3′‐terminal nucleotide to acquire both sufficient affinity and non‐selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double‐stranded DNA.

Chemiluminescent microtiter method for detecting PCR amplified HIV-1 DNA

A rapid and sensitive method for detecting HIV-1 DNA sequences amplified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigenin-labeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious amplification products. The presence of the digoxigenin-labeled amplified HIV target is then detected by anti-digoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets.

Colorimetric detection for PCR amplified HIV-1 DNA using magnetic beads

A rapid and nonradioactive detection method for polymerase chain reaction (PCR) amplified HIV-1 DNA was developed using a colorimetric detection system. Hybridization between biotin-labeled amplified targets and digoxigenin-capture probes occurs in solution followed by efficient and rapid capture onto streptavidin-magnetic beads. The presence of the digoxigenin-capture probe hybridized with biotin-labeled targets is then detected by antidigoxigenin-alkaline phosphatase conjugates using a colorimetric substrate. This approach is highly sensitive and can detect less than 10 HIV targets prior to PCR in approximately 50 min.