Hybridization of Peptide Nucleic Acid ² Tommi Ratilainen, ‡ Anders Holme ´n,* ,‡ Eimer Tuite, ‡ Gerald Haaima, § Leif Christensen, § Peter E. Nielsen, § and Bengt Norde ´n ‡ Department of Physical Chemistry, Chalmers UniVersity of Technology, S-412 96 Gothenburg, Sweden, and Center for Biomolecular Recognition, The Panum Institute, Department of Biochemistry B, BlegdamsVej 3c, DK-2200 N Copenhagen, Denmark ReceiVed April 17, 1998; ReVised Manuscript ReceiVed June 24, 1998 ABSTRACT: The thermodynamics of hybridization and the conformations of decameric mixed purine- pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 °C increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (∆T m )-12 °C) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA. Oligonucleotides and their mimics are becoming increas- ingly interesting for use as gene-targeted drugs and molecular biology tools (1, 2). Among these, peptide nucleic acids (PNAs) have been shown to have certain advantages compared to other analogues (3, 4). In PNA, the negatively charged backbone of DNA is completely exchanged for a neutral achiral pseudopeptide, composed of N-(2-amino- ethyl)glycine units, onto which the nucleobases are attached (Figure 1). A mixed-sequence PNA strand can hybridize with comple- mentary single-stranded DNA, RNA, or another PNA strand to form stable duplexes with high sequence-selectivity (3- 5). The thermal stabilities increase in the series DNA/DNA < PNA/DNA < PNA/PNA (5, 6). According to NMR experiments, PNA/DNA mixed-sequence duplexes adopt a structure possessing features of both A-form and B-form DNA (7), while oligopyrimidine PNAs with homopurine polynucleotides form triplex structures analogous to DNA triplexes, as concluded from flow linear dichroism and CD (8). The PNA/PNA duplex was inferred to be a B-DNA- like helical complex from CD measurements (9). However, X-ray diffraction studies have shown that a PNA/PNA duplex in a crystal adopts a unique P-form helix structure with ² Financial support from the Swedish Cancer Foundation (B.N.), the EU Biomed 2 Program (BMH4-CT96-0848, B.N.), and the Danish National Research Foundation (P.E.N.) is gratefully acknowledged. E.T. and A.H. thank the EU TMR program (ERBCHBGCT940548) and the Chemistry Division at Chalmers University of Technology, respectively, for research fellowships. * To whom correspondence should be addressed. E-mail: holmen@ phc.chalmers.se. Phone: +46-31-772 30 49. Fax: +46-31-772 38 58. ‡ Chalmers University of Technology. § The Panum Institute. FIGURE 1: Chemical structures of DNA and PNA. The deoxyribose phosphate ester backbone in DNA has been changed to N-(2- aminoethyl)glycine in PNA. 12331 Biochemistry 1998, 37, 12331-12342 S0006-2960(98)00872-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/14/1998