Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Abstract

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2′-O, 4′-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.     

Interactions of Archaeal Chromatin Proteins Alba1 and Alba2 with Nucleic Acids

Background

Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/Principal Findings

These two peptides were expressed and their interactions with various DNAs were studied using a combination of various experimental techniques: surface plasmon resonance, UV spectrophotometry, circular dichroism–spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/Significance

Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.     

Conformational Studies of Nucleic Acids: III. Empirical Multiple Correlation Functions for Nucleic Acid Torsion Angles

Abstract

There are seven significantly variable torsion angles in each monomer unit of a polynucleotide. Because of this, it is computationally infeasible to consider the energetics of all conformations available to a nucleic acid without the use of simplifications. In this paper, we develop functions suggested by and regression fit to crystallographic data which allow three of these torsion angles, α (03′-P-05′-C5′), δ (C5′-C4′-C3′-03′) and ε (C4′-C3′-03′-P), to be calculated as dependent variables of those remaining. Using these functions, the seven independent torsions are reduced to four, a reduction in complexity sufficient to allow an examination of the global conformational energetics of a nucleic acid for the remaining independent torsion angles. These functions are the first to quantitatively relate a dependent nucleic acid torsion angle to several different independent angles. In all three cases the data are fit reasonably well, and in one case, α, the fit is exceptionally good, lending support for the suitability of the functions in conformational searches. In addition, an examination of the most significant terms in each of the correlation functions allows insight into the physical basis for the correlations.     

Experimental Cyanine Red: A New Stain for Nucleic Acids and Acid Mucopolysaccharides

A new cationic dye, experimental cyanine red (du Pont), with an absorption maximum of 536 mμ and a pH of 2.9 in 0.5% aqueous solution, is shown to be suitable for staining nucleic acids and tissue materials presumed to contain acid mucopolysaccharides. Mammalian tissues fixed in 10% neutral buffered formalin or Bouin's fluid are dehydrated, embedded in paraffin, sectioned, mounted, deparaffinized, passed through ethanols to water, and stained for 3-30 min in 0.5% experimental cyanine red in water. Differentiation and dehydration in 3 changes (about 1 min each) of n-butanol is followed by clearing in xylene and mounting in resin.