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.

Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions. strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.

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