Mercurated polynucleotides. New probes for hybridization and selective polymer fractionation

Abstract

Polynucleotides containing covalently bound mercury atoms have been prepared by chemical or enzymatic syntheses and some of their physical and biochemical properties studied. The mercury substituents do not appear to alter significantly normal polynucleotide structure. Mercurated polymers function efficiently as templates for nucleic acid polymerases, they are fully susceptible to degradation by standard nucleases, and their denaturation and reannealing properties resemble those of the corresponding nonmercurated polymers. While the Tm's of DNA duplexes are lowered by extensive mercuration, the Tm's of DNA-RNA hybrids and RNA duplexes are either unaffected or elevated. Mercuration, as would be expected, greatly increases the buoyant density of both DNA and RNA. The introduction of as few as one mercury atom per 200 bases permits the selective and quantitative retention of the mercurated polymer probe (and associated nucleotide sequences) on columns of sulfhydryl-agarose. The use of mercurated nucleotides (as polymerase substrates) and oligonucleotides (as primers) in conjunction with sulfhydryl-agarose chromatography provides a simple and efficient method for the isolation of selected polynucleotide sequences, such as specific in vitro transcription products or terminal fragments of duplex DNA. Products absorbed to the affinity resin are readily recovered for further analysis by eluting with buffers containing mercaptoethanol. Although the mercury-carbon bond is somewhat thermolabile, mercurated polynucleotides are suitable as probes in low temperature hybridization studies.