Dodecamer d-AGATCTAGATCT and a Homologous Hairpin form Triplex in the Presence of Peptide REWER

Amrita Das,Vikas Rishi,Tapas Saha,Kunal B Roy,Faizan Ahmad,Claudine Mayer

doi:10.1371/journal.pone.0065010

Abstract

We have designed a dodecamer d-AGATCTAGATCT (RY12) with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer (RY28) that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5′- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin (RY28) melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase (melting of the duplex of RY12), second phase (melting of the triplex) and the third phase (melting of the hairpin) show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

Highlights

The considerable interest in DNA triple helix arose due to its role in human diseases [1,2] and its potential use in targeted gene therapy [3,4,5]
A prerequisite to an in vivo parallel triple helix between a single strand of DNA and the homologous duplex DNA is the localized unwinding of DNA double helix
REWER with a centrally placed tryptophan is suggested to do the same first by unfolding the DNA and enabling the invasion of third strand resulting in the formation of triple helix

Summary

Introduction

The considerable interest in DNA triple helix arose due to its role in human diseases [1,2] and its potential use in targeted gene therapy [3,4,5]. Several groups used designed deoxyribonucleotides to demonstrate that intermolecular triplexes where the third strand occupies the major groove of the duplex could form at sites that are purine-rich in one strand. In these triplexes the third strand can be either parallel or antiparallel. In PuNPuPy triplex type purine-rich third strand is hydrogen bonded to underlying purine strand of duplex in antiparallel orientation whereas pyrimidine-rich third strand in PyNPuPy triplex forms parallel triplex with purine strand of duplex. Contrary to the known antiparallel triplexes the third strand here is accommodated in the major groove of DNA duplex in parallel orientation of like strands with deoxyriboses in north conformation [9,10]. Several models of major groove associated parallel triplex structure have been proposed [11,12]

Methods

Results

Conclusion