Use of DNA molecules substituted with unnatural nucleotides to probe specific drug-DNA interactions

This chapter illustrates the application of DNA molecules substituted with unnatural nucleotides to probe specific drug-DNA interactions. The PCR-based strategy to incorporate modified bases into DNA has been optimized with the tyrTfragment, which is 160 bp long. The protocol should work as well with other DNA fragments of 100-250 base pairs (providing that they do not contain too many long runs of contiguous A · T or G · C pairs, which may reduce considerably the efficiency of incorporation of certain modified bases). The protocol has been successfully used in our laboratory to make analog-containing variants of many DNA fragments in this size range. Moreover, it is possible to effect several types of modifications concomitantly. A totally synthetic tyrT DNA species containing inosine· methylcytosine (I · M) and diaminopurine · uridine (D ·U) base pairs has been prepared in good yield by PCR. Although composed exclusively of I · M and D · U base pairs this DNA appears to bind to a histone octamer more tightly than does natural DNA and, surprisingly, its rotational orientation on the surface of the protein is not affected. DNA is highly adaptable and can evidently tolerate extensive modifications at the nucleotide level as well as at the level of its sugar–phosphate backbone. I, M, D, and U are only four representative examples of modified bases that can be incorporated into DNA via the PCR methodology presented here.