Polyamide Nucleic Acid-DNA Chimera Lacking the Phosphate Backbone Are Novel Primers for Polymerase Reaction Catalyzed by DNA Polymerases ² Hari S. Misra, Pradeep K. Pandey, ‡ Mukund J. Modak, Ravi Vinayak, § and Virendra N. Pandey* Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark, New Jersey 07103 ReceiVed June 24, 1997; ReVised Manuscript ReceiVed December 12, 1997 ABSTRACT: A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases. The analogue, (PNA) 19 -TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3′OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E. coli DNA polymerase I. The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases. The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone. Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3′OH terminus of the immobilized primer. PNA sequences were also recognized as template when annealed with a DNA primer. These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases. The efficient extension of (PNA) 19 -TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases. In 1991, Nielsen and his colleagues described the synthesis of a new type of DNA analogue which was named peptide nucleic acid (1). These analogues are structural homologs of DNA (Figure 1) with a peptide backbone of 2-amino- ethylglycine units to which purine and pyrimidine bases are linked (2, 3). Although PNAs 1 lack phosphate backbone, they hybridize with the complementary sequences in DNA or RNA. The oligomeric PNAs have been shown to recognize their complementary sequences even in the double- stranded DNA and are shown to form exceptionally stable complexes by strand displacement (1-3). These analogues can also form stable triplexes with dsDNA which are resistant to denaturation at temperatures where DNA duplexes of identical sequence exhibit melting (4). Despite the high stability of the PNA-DNA complexes, single base pair mismatches greatly reduce the melting temperature, suggest- ing that the PNA is able to recognize dsDNA in a sequence ² This research was supported in parts by a grant from the National Cancer Institute and the National Institute of General Medical Science (CA72821 to V.N.P.; GM36307 to M.J.M.). * Address correspondence to this author. Telephone: 973-972-5322. FAX: 973-972-5594. ‡ Predoctoral Fellow from Awadh University, Faizabad, India. § Present address: PE Applied Biosystems, 850 Lincoln Center Dr., Foster City, CA 94404. 1 Abbreviations: PNA, polyamide (peptide) nucleic acid; DiPEA, diisopropylethylamine; DMF, dimethylformamide; HBTU, benzotriazol- 1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HIV-1 RT, human immunodeficiency virus type 1 reverse transcriptase; IMAC, immobilized metal affinity chromatography; IDA-Sepharose, imino- diacetic acid-Sepharose; MuLV, murine leukemia virus; U5-PBS RNA template, 5′ non translated region of HIV-1 genomic RNA template containing the primer binding site (PBS); PBS-DNA template, HIV-1 genomic DNA template corresponding to the U5-PBS RNA sequence; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; DTT, dithiothreitol; TP, template primer; dATP, dGTP, dCTP, and dTTP, nucleoside triphosphate of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine, respectively. FIGURE 1: Structure for (PNA) 21 and (PNA) 19 -TpG-OH chimera. The sequences of (PNA) 21 and (PNA) 19 -TpG-OH chimera are shown in Chart 1. In the figure, b represents A, T, G, or C purine and pyrimidine bases linked with polyamide backbone and n represents the number of these bases in the molecule. (PNA) 21 has a single Lys amino acid at the end of (pseudo 3′ terminus) the sequence while the (PNA) 19 -TpG-OH chimeric molecule is comprised of 19 bases in the form of PNA followed by a normal dinucleotide TpG with G having a free 3′OH terminus. 1917 Biochemistry 1998, 37, 1917-1925 S0006-2960(97)01524-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/30/1998