Interactions of DNA binding ligands with PNA-DNA hybrids

Abstract

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA -DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxypsoralen and the A and A enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA -PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA -DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA -PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings. INTRODUCTIONThe interactions of double stranded DNA with small as well as with large molecules are often divided into contributions from the deoxyribose phosphate backbone and from the nucleobases themselves, although this segregation of binding/recognition components is not easily made. The main electrostatic component of the binding energy of cationic ligands is generally attributed to the negative phosphates of the DNA backbone, whereas the nucleobases contribute hydrophobic and dispersive bonding components but also considerable electrostatic components in terms of hydrogen and dipolar bonding (1,2). Specific interactions with the nucleobases naturally determine the sequence specificity exhibited by a ligand, for proteins they are also assisted by 'textured' properties such as helical pitch, indirectly determined by the base-sequence (3,4). However, the relative importance of electrostatic and hydrophobic contributions in the recognition/ binding process is far from clear, even for rather simple ligands such as typical intercalators and minor groove binders.We recently prepared a DNA analog termed PNA (peptide nucleic acid) containing an uncharged pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units to which the nucleobases are attached (Figure ...

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

“…This is noteworthy in light of previous investigations in which typical intercalators, such as ethidium bromide, 8-methoxypsoralen, and RuA C H T U N G T R E N N U N G (phen) 2 dppz 2 + , did not exhibit increases in fluorescence upon exposure to PNA·DNA duplexes. [41] However, analysis of the fluorescence titration data ( Figure 6C, D) revealed that TO-PRO1 binds PNA·DNA duplex 3·2 with lower affinity (K app = 7.3 AE 0.4 mm per base pair) than DNA·DNA duplex 1·2 (K app = 134 AE 20 mm per base pair) ( Table 1). Most remarkable were the results obtained with FIT-PNA complexes 4.…”

“…Previous investigations by NordØn showed that traditional intercalators, such as ethidium, methoxypsoralen, and ruthenium dipyridinophenazine complexes, failed to bind to PNA·DNA duplexes, whereas minor groove binders, such as DAPI (4',6-diamidino-2-phenylindole) and distamycin, showed modest binding affinities. [41] Armitage demonstrated that the minor groove of PNA·DNA duplexes provides a high-affinity template for the aggregation of the symmetrical cyanine dye DiSC 2 . [42,43] We have not attempted to resolve the stoichiometry of TO-PRO1·PNA·DNA complexes.…”

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

“…In the hybridized state, the dye can either fold back, binding to the PNA-DNA duplex (or PNA 2 -DNA triplex), or it can bind to the protruding single-stranded DNA. We think the latter dominates since intercalating dyes in general have very low affinity for PNA-DNA duplexes (and PNA 2 -DNA triplexes) (19), while TO has substantial affinity for single-stranded DNA (16). Still the spectroscopic properties of the probes in the hybridized state varied slightly, although the protruding sequences were the same in all constructs (Table 2).…”

Enzymatic reaction with unnatural substrates: DNA photolyase ( Escherichia coli ) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex

“…It appears that the only additional requirement for recognition by the enzyme is phosphate groups on the dimer-containing strand (3,4). For these reasons, we suspected that photolyase might retain its ability to recognize and repair thymine dimers contained on the DNA strand of a DNA͞PNA hybrid duplex (5)(6)(7)(8). We carried out a series of experiments to assess this possibility.…”