Use of SAM2 biotin capture membrane in microarrayed compound screening (muARCS) format for nucleic acid polymerization assays.
Microarrayed compound screening format (muARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the muARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM(2) biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM(2) membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM(2) membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in muARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of muARCS technology.
Xuei
Xiaoling
X
Global Pharmaceutical R&D, Abbott Laboratories, Abbott Park, IL, USA. xxuei@iupui.edu
David
Caroline A
CA
Middleton
Tim R
TR
Lim
Ben
B
Pithawalla
Ron
R
Chen
Chih-Ming
CM
Tripathi
Rakesh L
RL
Burns
David J
DJ
Warrior
Usha
U
D000480:Alkynes; D019380:Anti-HIV Agents / Q000494:pharmacology; D048588:Benzoxazines; D001710:Biotin / Q000737:chemistry / Q000494:pharmacology*; D019921:Biotinylation; D003521:Cyclopropanes; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D054303:HIV Reverse Transcriptase / Q000235:genetics; D020128:Inhibitory Concentration 50; D019829:Nevirapine / Q000494:pharmacology; D009696:Nucleic Acids / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010078:Oxazines / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D009696:Nucleic Acids; D010078:Oxazines; D012313:RNA; D001710:Biotin; D004247:DNA; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C098320:efavirenz