Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures.
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 (T(m)). 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 T(m) 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.
Shi
Xiaochun
X
Department of Biochemistry, University of Mississippi Medical Center, 2500 N. State St. Jackson, MS 39216-4505, USA.
Chaires
Jonathan B
JB
D001483:Base Sequence; D003630:Daunorubicin / Q000031:analogs & derivatives / Q000737:chemistry; D005821:Genetic Techniques; D008024:Ligands; D009691:Nucleic Acid Denaturation; D009696:Nucleic Acids / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011119:Polynucleotides / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D008024:Ligands; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D011119:Polynucleotides; C104426:WP 631; D003630:Daunorubicin