The use of peptide nucleic acids for in situ identification of human chromosomes.

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acid analogues, based on their peptide-like backbone. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double-stranded DNA, the unique physicochemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy, genome mapping, and mutation detection. Over the past few years, PNAs have been shown to be powerful tools in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, and spermatozoa, as well as on isolated oocytes and blastomeres. Multicolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful complement to FISH for in situ chromosomal investigation.     

The peptide nucleic acids (PNAs): introduction to a new class of probes for chromosomal investigation

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed. Peptide nucleic acids hybridize with complementary nucleic acids with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physicochemical properties of PNAs have led to the development of a large variety of biological research assays, and, over the last few years, PNAs have proved their powerful usefulness in genetic and cytogenetic diagnostic procedures. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic mutation or capturing nucleic acids. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, the in situ detection of several human chromosomes has been reported in various types of tissues.Communicated by E.A. Nigg     

The peptide nucleic acids (PNAs): a new generation of probes for genetic and cytogenetic analyses

Peptide nucleic acids (PNAs) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked. This structure gives PNAs the capacity to hybridize with high affinity and specificity to complementary sequences of DNA and RNA, and also confers remarkable resistance to DNAses and proteinases. The unique physico-chemical characteristics of PNAs have led to the development of a wide range of biological assays. Several exciting new applications of PNA technology have been published recently in genetics and cytogenetics. Also, PNA-based hybridization technology is developing rapidly within the field of in situ fluorescence hybridization, pointing out the great potential of PNA probes for chromosomal investigations.     

Information transfer from DNA to peptide nucleic acids by template-directed syntheses.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

Peptide nucleic acids (PNAs) antisense effect to bacterial growth and their application potentiality in biotechnology

Peptide nucleic acids (PNAs) are nucleic acid analogs having attractive properties such as quiet stability against nucleases and proteases, and they form strong complexes with complementary strands of DNA or RNA. Because of this attractive nature, PNA is often used in antisense technology to inhibit gene expression and microbial cell growth with high specificity. Many bacterial antisense or antiribosomal studies using PNA oligomers have been reported so far, and parameters to design effective antisense PNAs and to improve PNA cell entry for efficient inhibition of bacterial growth have been presented. However, there are still several obstacles such as low cellular uptake of PNA while applying antisense PNAs to a complex microbial community. On overcoming these problems, the PNA antisense technique might become a very attractive tool not only for controlling the microbial growth but also for further elucidating microbial ecology in complex microbial consortia. Here, we summarize and present recent studies on the development of antimicrobial PNAs targeting mRNAs and rRNAs. In addition, the application potentiality of antisense techniques in nonclinical biotechnology fields is discussed.     

Information transfer from peptide nucleic acids to RNA by template-directed syntheses.

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.     

PNA microarrays for hybridisation of unlabelled DNA samples

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.     

Peptide nucleic acids: deoxyribonucleic acid mimics with a peptide backbone

This tutorial briefly describes a new class of synthetic biopolymer, which is referred to as peptide nucleic acid (PNA). In PNA, individual nucleobases are linked to an achiral neutral peptide backbone. PNA exhibits the hybridization characteristic (e.g., Watson—Crick duplex formation) of DNA. The achiral peptide backbone provides similar interbase distances as natural DNA, and adequate flexibility to permit base pair interactions with complementary RNA or DNA strands. Several potential applications of PNA oligomers in biotechnology are suggested. These include the use of PNAs as a probe for specific recognition of a DNA or RNA sequence selective, purification of nucleic acids via designed high affinity binding to PNA, screening for DNA mutations, and as possible therapeutic agents.     

Conjugates of PNA with naphthalene diimide derivatives having a broad range of DNA affinities

Peptide nucleic acids (PNAs) are neutral DNA analogues, which bind single-stranded DNA (ssDNA) strongly and with high sequence specificity. However, binding efficiency is dependent on the purine content of the PNA strand. This property make more difficult application of PNA as hybridization probes in, e.g., PNA chips, since at a set temperature the hybridization of a fraction of the DNA targets to the PNA probes does not obey Watson-Crick binding rules. The polypurine PNAs, for example, bind the mismatch containing DNA targets stronger, than the pyrimidine rich PNAs their fully complementary targets. Herein we show that PNA-DNA binding efficiency can be finely tuned by the conjugation of derivatives of naphthalene diimide (NADI) to the N-terminus of PNA using polyamide linkers of different lengths. This approach can potentially be used for the design of PNA probes, which bind their DNA targets with similar affinity independently of the PNA sequence.     

PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.     

Applications of peptide nucleic acids

Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently. A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs. In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and antisense activity has also been demonstrated in Escherichia coli. PNA hybridization technology has developed rapidly within in situ hybridization, and exciting new methods based on MALDI-TOF detection have also been presented.     

Role of chirality and optical purity in nucleic acid recognition by PNA and PNA analogs

Peptide nucleic acids are DNA mimics able to form duplexes with complementary DNA or RNA strands of remarkable affinity and selectivity. Oligopyrimidine PNA can displace one strand of dsDNA by forming PNA(2):DNA triplexes of very high stability. Many PNA analogs have been described in recent years, in particular, chiral PNA analogs. In the present article the results obtained recently using PNA derived from N-aminoethylamino acids 7 are illustrated. In particular, the dependence of optical purity on synthetic methodologies and a rationale for the observed effects of chirality on DNA binding ability is proposed. Chirality as a tool for improving sequence selectivity is also described. PNA analogs derived from D- or L-ornithine 8 were also found to be subjected to epimerization during solid phase synthesis. Modification of the coupling conditions or the use of a submonomeric strategy greatly reduced epimerization. The optically pure oligothymine PNAs 8 were found to bind to RNA by forming triplexes of unusual CD spectra. The melting curves of these adducts presented two transitions, suggesting a conformational change followed by melting at high temperature.