SYNTHETIC POLYNUCLEOTIDE TEMPLATES FOR CHARACTERIZING SEQUENCE-SELECTIVE SMALL MOLECULE NUCLEIC ACID INTERACTIONS/

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 v arious forms of nucleic acids. The target sequence comprehensively rep resents all base quartet recognition sites in a minimal length sequenc e. Minimal length target sequences were found to be 144 nt long. One s uch target sequence was divided into two parts. One strand of each par t was chemically synthesized and the complementary strands were genera ted using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/-) vectors (Promega, Inc.). The cloned target sequenc e can be used directly in double-stranded DNA form. Alternatively, fea tures of the plasmid vector allow expression of the target sequences a s 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 qu artets and the potential for secondary structure formation in single-s tranded forms. (C) 1997 Academic Press, Inc.