Methodology article

Specificity of DNA triple helix formation analyzed by a FRET assay

Sabine Reither^1,2 and Albert Jeltsch¹

¹Institut für Biochemie, FB8 Justus-Liebig-Universität Heinrich-Buff-Ring 58 35392 Giessen Germany

²present address: Institut für Genetik, Universität des Saarlandes Postfach 151150 66041 Saarbrücken Germany

author email corresponding author email

BMC Biochemistry 2002, 3:27doi:10.1186/1471-2091-3-27

Published:	12 September 2002

Abstract

Background

A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.

Results

We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 × 10⁵ and 2.3 × 10⁶ M^-1. Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg²⁺ions whereas it is inhibited by K⁺ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 10⁴ and 8.1 × 10⁴ min^-1 M^-1. Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation.

Conclusion

We have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.