TRIPLE-HELIX FORMATION ON PLASMID DNA DETERMINED BY A SIZE-EXCLUSION CHROMATOGRAPHIC METHOD

Triple-helix forming oligodeoxynucleotides are receiving considerable attention due to their potential applications for the inhibition of sp ecific genes in vivo. However, their development is impaired by the la ck of triple helix formation under physiological conditions. It is thu s crucial to be able to quantitatively assay triple helix formation of various oligodeoxynucleotides on different target sequences. Usual me thods to detect triple helix formation are restricted under the experi mental conditions that can be studied. In addition, quantitative techn iques are limited, We present a novel method for rapid detection and q uantification of triple helix formation between an oligodeoxynucleotid e and a plasmid carrying a target sequence. The oligodeoxynucleotide w as radiolabeled and, after incubation with the target plasmid, the unb ound oligodeoxynucleotide was separated from the mixture of plasmids a nd plasmid-bound oligodeoxynucleotides by rapid gel filtration spun co lumns. The formation of a triple helix between a target plasmid and se veral oligodeoxynucleotides was demonstrated and compared. Temperature , sequence and ionic dependencies, and kinetics of association were an alyzed. This new technique can be used under a variety of conditions a nd should allow the rapid determination of optimal conditions required for triple helix formation, as web as the easy selection of an oligod eoxynucleotide that specifically binds with the highest affinity to a target double-stranded sequence. (C) 1997 Academic Press.