PCR-based development of DNA substrates containing modified bases: an efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins.

DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of sequence-selective ligands, both small and large. Footprinting patterns reveal that the binding sites for AT- or GC-specific antibiotics (distamycin or mithramycin, respectively) are completely changed in the modified DNAs, as expected for direct sequence readout involving contact with the purine 2-amino group. However, we also find large changes in the binding of HMG-D, a member of the HMG-1 family of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformability of the double helix. This interpretation is confirmed by the finding that deoxyuridine-containing poly- and oligonucleotides, which lack the exocyclic methyl group of thymidine in the major groove, interact 5-10 times more strongly with HMG-D than do their counterparts containing natural nucleotides.