Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic acid (PNA) is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one another to form a helical duplex. There is a seeding of preferred chirality which is induced by the presence of an L- (or D-) lysine residue attached at the carboxy terminus of the PNA strand. These results indicate that a (deoxy)ribose phosphate backbone is not an essential requirement for the formation of double helical DNA-like structures in solution.

Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA−DNA duplex and the corresponding DNA−DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson−Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA−DNA, PNA−PNA, and DNA−DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3‘) and parallel (N/5‘) PNA−DNA duplexes whereas they stabilize the DNA−DNA duplex. Studies on the effect of monovalent salt such as NaCl on T m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T m of the N/3‘ PNA−DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA−PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3‘ PNA−DNA compared to the DNA−DNA duplex (ΔΔG ∼ −7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA−DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T m were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3‘ PNA−DNA and PNA−PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.

Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

Complementary peptide nucleic acids (PNA) form Watson-Crick base-paired helical duplexes. The preferred helicity of such a duplex is determined by a chiral amino acid attached to the C-terminus. We here show that the induced helicity, as measured by circular dichroism (CD), is drastically dependent on the nucleobase sequence proximal to the chiral center. Chemically linked PNA tetramer duplexes of all 16 combinations of the two bases proximal to a carboxy terminal lysine residue were studied by CD. We conclude that the base next to the chiral center must be either a guanine or a cytosine for efficient stabilization of one helical sense. In case of cytosine, the subsequent base should preferably be a purine. We also show that the side chain properties of the C-terminal amino acid influence the resulting sense of helicity. The propagation length of induced chirality in PNA duplexes is found to be around 10 base pairs. Theoretical calculations of the circular dichroism for B-DNA, using the quantum mechanical matrix method of Schellman, give spectra in reasonable agreement with those found experimentally for PNA duplexes. The rate of helix conversion of the duplexes shows first-order kinetics with a rate constant in the range of minutes. Shorter duplexes are found to have lower activation energy and larger negative activation entropy for helix conversion, in agreement with a conversion mechanism in which a perfect helix is switched to the opposite handedness.