DNA triple helix formation at target sites containing several pyrimidine interruptions: Stabilization by protonated cytosine or 5-(1-propargylamino)dU

Gowers, Darren M., Bijapur, Jeevan, Brown, Tom and Fox, Keith R. (1999) DNA triple helix formation at target sites containing several pyrimidine interruptions: Stabilization by protonated cytosine or 5-(1-propargylamino)dU. Biochemistry, 38 (41), 13747-13758. (doi:10.1021/bi9911637).

Abstract

DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G·TA triplets, while CG base pairs are targeted with thymine, forming T·CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1- propargylamino)dU (U(P)). For the target sequence (AAAT)₅AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex- binding ligand, though stable triplexes were detected at the target site (AAAAAT)₃AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)₅AA and (ATGA)₅AA. In contrast, placing C⁺·GC triplets on the 5'-side of G·TA, using the target (AGTA)₅TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)₅TT forms a more stable complex with target (AAAT)₅AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)₅AA is improved by using the modified oligonucleotide (TCGU(P))₅TT, generating a complex in which the charged triplets C⁺·GC and U(P)·AT alternate with uncharged triplets. In contrast, placing U(P)·AT triplets adjacent to C⁺·GC, using the third strand oligonucleotide (U(P)CGT)₅TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))₅TT, produces a complex with intermediate stability. It appears that, although adjacent U(P)·AT triplets form stable triplexes, placing U(P)·AT adjacent to C⁺·GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)₃AA were only detected in the presence of a triplex- binding ligand. Placing C⁺·GC on the 5'-side of T·CG triplets also reduces triplex formation, while a 3'-C⁺·GC produces complexes with increased stability.

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Contributors

Author: Keith R. Fox

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