Strand-invasion of duplex DNA by peptide nucleic acid oligomers.

Abstract

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone ofthe PNA could permit binding to a DNA target in an orientationindependent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity-that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNADNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase lI-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA-DNA complex was attributed to a bend in the DNA at or near the D-loop.The inhibition of gene expression mediated by oligodeoxynucleotides is a powerful research tool that has been exploited to study the function of specific gene products in complex biological pathways (for reviews, see refs. 1 and 2). Although technically challenging, inhibiting gene expression at the level of transcriptional initiation and/or RNA polymerase elongation (antigene approaches) may represent the most fundamental means of ablating gene function. Two mechanisms by which gene-specific inhibition of transcription could be achieved in vivo are oligomer binding in the major groove of duplex DNA to form a triple helix (3-5) or by strand-invasion of duplex DNA, resulting in the formation of a stable D-loop [using peptide nucleic acids (PNAs) (6-10)]. In the case of triplex formation, recognition is mediated by Hoogsteen base pairing (11). The nonphysiologic conditions necessary to promote third-strand binding of an oligomer to a duplex target has largely precluded the use of this approach for in vivo studies. However, the requirement for low pH or specific ionic conditions to stabilize cytosine protonation (12, 13) has recently been obviated by pH-independent cytosine surrogates (14, 15).PNAs consist of achiral monomer subunits derived from N-ethylaminoglycine bearing appendent nucleoside heterocyclic bases, which are coupled by standard methods of amide bond formation. We and others (6, 9, 10) have shown that thymidine-rich PNAs recognize complimentary sequences in duplex DNA by strand-invasion, which results inThe publication costs of this article were defrayed in part by page charge payment. This article must therefore ...

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