A family of unusual DNA structures has been discovered in segments with predominantly purines in one strand (pur.pyr sequences). These sequences are overrepresented in eukaryotic DNA and have been mapped near genes and recombination hot spots. When cloned into recombinant plasmids, many pur.pyr sequences are reactive to chemical and enzymic probes that are generally specific for single-stranded DNA. An intramolecular triplex is adopted by mirror repeats of G's and A's. Other non-B DNA structures adopted by similar sequences remain to be fully clarified but may be a family of related conformations. It is likely that these unorthodox structures play an important role in the function of the eukaryotic genome.
Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 15-mer PNA to the transcribed strand of a G-free transcription cassette caused 90 to 100 percent site-specific termination of pol II transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA.RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in beta-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.
A series of inserts with ollgopurineoligopyrimidine mirror repeat sequences was investigated at the base pair level with specific chemical probes (OS04 and diethylpyrocarbonate) to evaluate the in vitro existence of intramolecular triplexes. Two (Fig. 1). Hence, the TOAST (or U) and CG-C arrangements provided substantial specificity for the interactions. Formation of two hydrogen bonds between (G). and cytosine residues in the third strand requires hemi-protonation of cytosines. However, the pH of deoxycytosine residues in polynucleotides is above pH 7 (26, 27) and hence is consistent with physiological conditions. Herein, we prepared a family of inserts designed specifically to test the existence of the highly unorthodox triplex model in plasmids. Certain base pair changes were instituted to elicit instability in the putative triplex at specific loci some MATERIALS AND METHODSPlasmids. pRW791 was made by filling in the EcoRI site of pRW790 (15,28). Oligonucleotides were synthesized and cloned into the BamHI site of pRW790 (or pRW791 for the pRW1704 insert) as described (2,15,16,22,28,29). The inserts were synthesized with GATCC on the 5' end and a guanine on the 3' end, except for the pRW1704 insert, which contained a TTCG on the 3' end of the purine strand. The insert in pRW1402 was found when screening transformants for the insert of pRW1404.Chemical Modifications. Chemical modifications of the plasmids were as described (2). For the reactions at elevated temperatures, the mixtures were preincubated for 15 min at 250C and then 15 min at the indicated temperatures. The reactions were stopped by placing the containers on ice, and the subsequent workup was as described (2). 6292The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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 ...
The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.
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