Synthetic Polynucleotide Templates for Characterizing Sequence-Selective Small Molecule/Nucleic Acid Interactions

Abstract

A synthetic polynucleotide sequence was developed and cloned to serve as a target in footprinting and other assays designed to characterize the sequence-selective binding of drugs and other small molecules to various forms of nucleic acids. The target sequence comprehensively represents all base quartet recognition sites in a minimal length sequence. Minimal length target sequences were found to be 144 nt long. One such target sequence was divided into two parts. One strand of each part was chemically synthesized and the complementary strands were generated using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/−) vectors (Promega, Inc.). The cloned target sequence can be used directly in double-stranded DNA form. Alternatively, features of the plasmid vector allow expression of the target sequences as single-stranded DNA or RNA or as RNA/DNA or RNA/RNA duplexes. These cloned target sequences designed for high information content overcome limitations to the use of natural DNA sequences for footprinting and related experiments arising from the unequal representation of base quartets and the potential for secondary structure formation in single-stranded forms.