Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

Abstract

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (Tm). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the Tm is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds.

Drug Binding to DNA⋅RNA Hybrid Structures

“…The assay in its current form typically includes both poly(dA)·poly(U) and poly(A)·poly(dT) hybrids alongside the counterpart pure DNA and pure RNA duplexes. The melting of mixtures assay (26) is an extension of the well-established DNA thermal denaturation assay (27, 28) that detects the effects of added ligand on thermal denaturation of four duplexes simultaneously. It is valuable for rapidly assessing binding preferences amongst DNA, RNA and hybrid duplexes; for the hybrid systems it also provides evidence of selectivity between dA·U and A·dT sequences.…”

“…Qualitatively, an increase in the melting temperature for a nucleic acids structure provides unambiguous evidence for binding. A recent extension of the thermal denaturation assay resulted from the simple expediency of making a mixture of different nucleic acid structures that could be distinguished by their melting temperatures(26). Addition of low binding ratios of a test ligand altered the melting temperature of the nucleic acid structure to which it bound most avidly, providing a simple, rapid evaluation of its structural preference.…”

“…Addition of low binding ratios of a test ligand altered the melting temperature of the nucleic acid structure to which it bound most avidly, providing a simple, rapid evaluation of its structural preference. A mixture containing DNA, RNA, and two DNA·RNA hybrid forms was described and tested (26), and a protocol for that preparation and use of that mixture will be described here.…”

A modular design for minor groove binding and recognition of mixed base pair sequences of DNA

“…24,25 Binding to DNA gives an increase in the melting temperature that is related to binding affinity. Hairpin DNA oligomers, which have monomolecular melting transitions, were chosen for the screen.…”

Determination of in silico rules for predicting small molecule binding behavior to nucleic acids in vitro.

“…To accomplish this, competition dialysis was employed because of its extensive use to determine the selectivity and affinity of a small molecule for single stranded, duplex, triplex and quadruplex nucleic acid targets [7,10,130,164,[172][173][174][175][176][177][178][179]. The advantage of competition dialysis is that it is not limited to the target sequence, or a simple comparison with one other form of DNA, but with as many nucleic acid forms as are included in the assay.…”