Methods of Using Phosphoramidation Reaction for Conjugation-Labeling Nucleic Acids

摘要

Methods are provided to label nucleic acids through two-step phosphoramidation reactions with regiospecificity and high sensitivity. The methods can be widely used for synthesizing conjugates of nucleic acids. Through the two-step phosphoramidation reactions, nucleic acids are directly or indirectly labeled for easier and more efficient applications. The present invention also uses fluorescence quenching and DNA melting profile studies to demonstrate that the nucleic acids modified through the two-step phosphoramidation reactions preserve intrinsic base-pairing specificity and bestow regioselectivity in nucleic acids with high yield. Thus, the present invention has significant contribution to basic nucleic acid research; and, in the future, can be applied to effectively and quantitatively detect and measure nucleic acids inside and outside living organisms.

主权项

1. A method of modifying a nucleic acid with a label molecule through a phosphoramidation reaction, the method modifying said label molecules on said nucleic acids through direct coupling and comprising steps of:
(a1) dissolving a nucleic acid and an activating reagent in a first buffer to be reacted for 70˜100 minutes (min) to obtain a solution, said nucleic acids having phosphate at the 5′ end; (b1) processing purification and precipitation with ethanol to obtain an intermediate product; and (c1) dissolving said intermediate product in a second buffer to be added with a nucleophilic reagent to be reacted for 2.5˜3 hours (hr) to obtain a final product, wherein said second buffer contains ethylene-diaminetetraacetic acid (EDTA); wherein said nucleophilic reagent is a fluorophore; said fluorophore is a fluorescent molecule containing amine; and said fluorophore is selected from a group consisting of lissamine rhodamine B ethylenediamine (LRBE) and 5-(2-aminoethylamino)-1-naphthalenesulfonic acid sodium salt (EDANS).