Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.     

Automated Chemical Synthesis of PNA and PNA-DNA Chimera on a Nucleic Acid Synthesizer

Abstract

Automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.     

Synthesis and DNA binding properties of DNA-PNA chimeras

A systematic study to evaluate the ability of 5'-DNA-3'-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3'-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.     

Targeting duplex DNA with DNA‐PNA chimeras? Physico‐chemical characterization of a triplex DNA‐PNA/DNA/DNA

Targeting double-stranded DNA with homopyrimidine PNAs results in strand displacement complexes PNA/DNA/PNA rather than PNA/DNA/DNA triplex structures. Not much is known about the binding properties of DNA-PNA chimeras. A 16-mer 5'-DNA-3'-p-(N)PNA(C) has been investigated for its ability to hybridize a complementary duplex DNA by DSC, CD, and molecular modeling studies. The obtained results showed the formation of a triplex structure having similar, if not slightly higher, stability compared to the same all-DNA complex.     

Effect of temperature and ionic strength on the dissociation kinetics and lifetime of PNA-DNA triplexes

Dissociation kinetics of triplexes formed by molecules of peptide nucleic acid (PNA) and DNA have been studied. The complexes consisted of oligomeric PNA containing 10 thymine bases and the dA(10) target incorporated in single-stranded (ssDNA) or double-stranded DNA (dsDNA). Their dissociation was followed by means of the gel mobility shift assay at various temperatures and sodium ion concentrations. In all experiments, the dissociation kinetics of triplexes were exponential; the effective lifetime of a triplex, tau, depended on temperature in accordance with the Arrhenius law. The tau values for T(10) PNA complexes with ss- and dsDNA were equal within the accuracy of experiments. The activation energy, U, value for T(10) PNA-DNA complexes did not change when the NaCl concentration was increased from 50 to 200 or 600 mM. Conversely, the tau values decreased with the increase in NaCl concentration. The equal lifetimes of the T(10) PNA-DNA triplexes containing ss- and dsDNA suggest that the loop formed in dsDNA does not noticeably affect the triplex structure. The decrease in the triplex lifetime tau with an increase in ionic strength was accounted for by the fact that the PNA backbone is neutral. The lack of relationship between the activation energy of dissociation and salt concentration suggests that the dissociation enthalpy does not depend on the ionic strength. Thus, the effect of ionic strength on the lifetime is entropic by its nature. Contrary to this, for complexes of ssDNA with bis-PNA 1743, which also consists of 10 thymine bases but contains 2 additional positive charges inside the sequence in 1 of the PNA arms, an increase of the dissociation enthalpy at low salt concentration was observed. We suggest that this effect is a result of a direct electrostatic interaction of the positive charges of the PNA with the DNA backbone. Finally, our results allow an estimate of the lifetime of a 10-mer triplex invasion complex in dsDNA at 37 degrees C in excess of several hundred days.     

肽核酸(peptide nucleic acid,PNA)阵列

肽核酸(PNA)以N—(2—氨基乙基)甘氨酸替代DNA分子中的磷酸戊糖骨架。它能特异性地识别与DNA、RNA所形成的杂交体。PNA—DNA、PNA—RNA的热稳定性要比相应的DNA—DNA、DNA—RNA高,而且PNA识别单碱基的能力强于DNA和RNA,使之在微阵列,尤其是SNP检测领域有着广泛的应用前景。本文简述了PNA阵列从探针设计、阵列合成、杂交和检测的全过程。     

Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.     

Synthesis and DNA Binding Properties of DNA-PNA Chimeras

Abstract

A systematic study to evaluate the ability of 5′-DNA-3′-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3′-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.     

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.     

Synthesis, biophysical, and biochemical properties of PNA-DNA chimeras

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T20-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA20 and their hydrolyses by 3'- and 5'-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

PNA-DNA chimeras forming quadruplex structures

1H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized 5'TGGG3'-t (1) and 5'TGG3'-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]4. 1H-NMR spectrum for 5'TGG3'-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.     

Peptide nucleic acid (PNA) amphiphiles: synthesis, self-assembly, and duplex stability

Peptide amphiphiles comprising a class of conjugates of peptide nucleic acid (PNA), natural amino acids, and n-alkanes were synthesized and studied. These PNA amphiphiles (PNAA) self-assemble at concentrations between 10 and 50 muM and exhibit water solubilities above 500 muM. The highly specific, stable DNA binding properties of PNAs are preserved by these modifications, with no significant differences between the thermodynamics of DNA binding of the PNA peptide and the PNA amphiphile. Proper solubilization of the PNAA required the attachment of (Lys)(2) and (Glu)(4) peptides to PNAs, which affected the PNAA-DNA duplex stability by electrostatic interactions between these charged amino acids and the negatively charged DNA backbone. These electrostatic effects did not affect the specificity of DNA binding, however. Electrostatic effects are screened with added salt, in a manner consistent with previous studies of PNA-DNA duplex stability and predictions from a charged-cylinder model for the duplex.     

肽核酸探针在微生物诊断领域的应用进展

肽核酸(Polyamide nucleic acid,PNA)是以中性酰胺键为骨架的脱氧核糖核酸(DNA)结构类似物,它可以特异性地与DNA杂交,且具有极高的生物稳定性.相比较传统的DNA探针技术,肽核酸探针以其特殊的结构和性质在食品、环境及临床等微生物快速诊断领域显示出独特的优势.就肽核酸探针在微生物诊断方面的应用进展做简要综述.     

Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA₂–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA₂ triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA₂–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA₂–PNA triplex is significantly more stable than a DNA₂–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.     

肽核酸在分子生物学技术中的应用

肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同。PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构。与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点。简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望。     

Induction of RNase H activity by Arabinose-peptide nucleic acid chimeras

We report the syntheses of chimeras of peptide nucleic acid (PNA) with DNA and 2'-deoxy 2'-fluoroarabinonucleic acid (2'-FANA). Chimeric oligomers possessing a single central PNA insert were capable of forming hybrid duplexes with complementary RNA, although with diminished thermal stability in comparison to the unmodified oligomers. We subsequently determined the ability of the DNA and 2'-FANA oligomers of mixed-base composition to elicit human RNase H1 degradation of complementary RNA that was either unstructured or as a hairpin. In the case of the more rigid FANA strand, a PNA insert led to a higher ability of the chimera to direct the degradation of both types of RNA targets. Generally, the enhancement observed was greater for a butanediol linker than for a more rigid PNA linker. Along with previous work, these studies suggest that the general flexibility associated with an acyclic insert (e.g., butyl vs PNA)--and not necessarily the presence of local structural imperfections in the heteroduplex--is beneficial for RNase H1 activity. As well, there are implications to the charge nature of non-nucleotide inserts (neutral vs negative) and their ability to maintain RNase H activity that may serve to direct further design considerations. Together, these studies support the notion that flexibility of antisense oligonucleotide (AON)/RNA hybrids is essential for high RNase H catalysis, in which an enzyme-induced altered trajectory of the bound AON/RNA substrate could facilitate optimal interaction with the catalytic site of RNase H.     

A short phosphodiester window is sufficient to direct RNase H-dependent RNA cleavage by antisense peptide nucleic acid

The potential pharmacologic benefits of using peptide nucleic acid (PNA) as an antisense agent are tempered by its incapacity to activate RNase H. The mixed backbone oligonucleotide (ON) (or gapmer) approach, in which a short internal window of RNAse H-competent residues is embedded within an RNase H-incompetent ON has not been applied previously to PNA because PNA and DNA hybridize to RNA with very different helical structures, creating structural perturbations at the two PNA-DNA junctions. It is demonstrated here for the first time that a short internal phosphodiester window within a PNA is sufficient to evoke the RNase H-dependent cleavage of a targeted RNA and to abrogate translation elongation in a well-characterized in vitro assay.     

Alpha-L-RNA (alpha-L-ribo configured RNA): synthesis and RNA-selective hybridization of alpha-L-RNA/alpha-L-LNA chimera

Synthesis of the novel alpha-L-ribofuranosyl phosphoramidite derivative was accomplished via the alpha-L-ribofuranosyl thymine nucleoside. Amidite was used in automated syntheses of chimeric oligonucleotides composed of mixtures of the novel alpha-L-RNA nucleotide monomer ((alphaL)T, alpha-L-ribo configured RNA), and DNA, LNA (T(L), locked nucleic acid) or alpha-L-LNA ((alphaL)T(L), alpha-L-ribo configured locked nucleic acid) nucleotide monomers. For alpha-L-RNA/DNA and alpha-L-RNA/alpha-L-LNA chimeras, RNA-selective hybridization was obtained, for alpha-L-RNA/alpha-L-LNA chimera we found increased binding affinity compared to the corresponding DNA:RNA reference duplex. In addition, alpha-L-RNA/alpha-L-LNA chimera displayed significant stabilization towards 3'-exonucleolytic degradation. These results indicate that alpha-L-RNA/alpha-L-LNA chimeras deserve further evaluation as antisense molecules.