Novel Biolabeling Methods and Reagents

DESCRIPTION (provided by applicant): A set of novel ATP-fluorescent-dye molecules and ATP-biotin molecules has been developed for the enzymatic, 5'-end-labeling of an oligonucleotide. This preliminary set is composed of unique linkers and either fluorescent dye or biotin combinations attached at the terminal (gamma)-phosphate of ATP. Enzymatic transfer of the labeled gamma-phosphate is mediated by T4 Polynucleotide Kinase (T4 PNK) resulting in the specific 5' end-labeling of a DMA or RNA molecule. We have identified labeling of RNA for use in microarray analyses as the first application for this novel technology, since current methods for labeling microarray targets are sequenced biased, inconsistent, and labor intensive. Our method requires minimal handling and results in exclusive labeling at the 5' end of the molecule resulting in minimal impact on hybridization. The goal of the Phase I project is to examine the labeling efficiencies of a library of labeled ATP variants and define the critical threshold for obtaining robust and reproducible signal intensities from labeled-targets hybridized to a microarray. A group of commonly-used, commercially-available fluorescent dyes along with biotin, have been selected for microarray testing. The chemical procedure for synthesizing each of the ATP-linker- molecule combinations has been developed in conjunction with a screening strategy that examines fluorescent-labeling and biotin-labeling of nucleic acids. Phase II of the project will involve mutagenizing key residues within T4 PNK to enhance its catalytic activity with the optimized Phase I library of ATP-molecules.