(1) Whitehead Institute for Biomedical Research, Dept. of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, and (2) Millipore Corporation, Core R & D, Specialty Chemistry Group, 75A Wiggins Ave., Bedford, MA 01 730
In 1991, Nielsen, Egholm, Berg, and Buchardt (I) reported the synthesis of peptide nucleic acids (PNA's), a new, completely artificial DNA/RNA analog. The most impressive property of PNA's are their ability to form extremely stable complexes with complementary DNA oligomers. A 10-mer PNA:DNA complex denatures at a melting temperature of 73deg.C. This stability suggests PNA's are superior reagents in anti-sense and anti-gene applications and creates other uses for which sequence specific but thermally stable complexes are required. In this article we will describe the structure of PNA monomers and oligomers, summarize key aspects of the synthesis, and describe some properties and applications of PNA:DNA complexes.
PNA's - Structure and Synthesis
The PNA monomer is 2-aminoethyl glycine linked by a methylenecarbonyl linkage to one of the four bases (adenine, guanine, thymine, or cytosine) found in DNA. Like amino acids, PNA monomers have amino and carboxyl termini. Unlike nucleotides, PNA's lack pentose sugar phosphate groups. The general structure of the PNA monomer is shown in Fig. 1.
PNA monomers are linked by peptide bonds into a single chain oligomer. By convention, the PNA oligomer is depicted like a peptide with its N-terminus at the first position (Fig. 2). However, this end corresponds to the 3' end of a DNA or RNA strand. Hence, the N-terminus of a PNA hybridizes to the 5'-end of complementary single-stranded DNA.
Thus, unlike the 5' to 3' convention in writing nucleic acid sequences, PNA sequences are usually written from 3' to 5'. Although PNA oligomers share similar structures with peptides and oligonucleotides, the non-standard backbone confers resistance to degradation by proteases and nucleases.
PNA monomers are easily synthesized into oligomers as long as 20 bases using protocols for standard peptide synthesis. The L monomers are supplied by Millipore as Boc-benzyloxycarbonylprotected derivatives and are soluble in DMF/pyridine but only sparingly soluble in 112O. Monomers are coupled in DMF/pyridine containing HBTU and a tertiary amine. Typical coupling yields are >95%. Synthesis is completed by TFMSA cleavage of the oligomer from the resin. The oligomer is purified by reverse phase HPLC. Although the monomers exhibit poor solubility, the oligomers are very soluble (10-40 mg/ml) in H2O, especially if exocyclic amines are present. The mass of the oligomer, as estimated by matrix-assisted laser desorption mass spectrometry, is used to confirm the synthesis.
PNA:DNA triplexes
In published reports (1,2) the Tm of a 6-mer PNA T:DNA dA mixture, measured by the midpoint in the hypochromic shift, is 31deg.C. In comparison, the Tm of the corresponding DNA:DNA duplex is 15deg.C. Longer PNA's exhibit higher Tm's. A 10-mer PNA I :DNA A mixture melted at 73deg.C. T he high thermal stability results from the formation of a triple-stranded complex having a 2PNA: 1 DNA stoichiometry. The complex is hypothesized to be a PNA:DNA duplex maintained by standard Watson-Crick base pair interactions. A second PNA strand lies in the major groove of the duplex where it makes Hoogstein hydrogen bonds to bases in the PNA:DNA duplex.
The same trends have now been replicated with PNA's of mixed sequences, which are also presumed to form triplexes in solution. A complicating factor, however, is that the Tm of any PNA:DNA complex is influenced by the sequence. Unlike DNA or RNA duplexes, it does not appear to be possible to predict the Tm deg.f a PNA:DNA mixture. Single base mismatches lower the Tm by approximately 1 5deg.C but the magnitude of the decrease depends on the position and the base.
PNA Applications
The high thermal stability and resistance to proteases and nuclease make PNA's ideal reagents in an anti-gene or anti-sense study. Resistance to degradation should increase the half-life ol the reagent in the cell or in cell culture media and simultaneously decrease the dose required for inhibition. An area of active investigation is focused on methods to introduce anti-sense reagents into a cell and to target the reagent to a particular organ in an animal. One report documents that expression of SV-40 large T antigen was inhibited when a sequence specific 1 5-mer or 20-mer PNA was microinjected into the nuclei of cells (3). Previously developed methods for introducing nucleic acid analogs into cells should be at least equally successful for PNA applications. PNA's offer the additional advantage that PNA-peptide chimeras can be synthesized. Such combinations may offer additional mechanisms to target PNA's to specific locations.
We are collaborating to use PNA's as markers in DNA mapping projects. Much of the current effort in the Human Genome Project is to construct physical maps based on the locations of STS's. Currently, STS's are mapped by PCR-based methods to genomic sequences in YAC libraries. PNA's may simplify and speed PCR-based screening methods. In addition, the locations of fluorescent PNA's may be mapped directly onto genomic DNA using fluorescence in situ hybridization. The thermal stability of short PNA's offer the possibility of mapping transcription factor binding sites, regulatory sites in DNA, restriction sites, and short repetitive sequences. To improve the precision of the maps we are straightening DNA molecules with optical tweezers.
The PNA Future
The PNA literature numbers less than 20 papers. This is obviously an early stage in studies using PNA oligomers. Much work in basic research is required to understand the physical rules which govern PNA-DNA interactions. Nevertheless, the unique and superior properties of this polymer predict widespread applications in biology, medicine, and industry.
1. Nielsen et al., (1991) Science 254, 1497.
2. Eghom et al., (1992) J. Am. Chem. Soc. 114, 1895.
3. llanvey et al., (1992) Science 258, 1481.
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