Aromatic π-π interactions are important  in the stabilization of helical nucleic acid structures. Recently, we have discovered that pyrene nucleotide is a selective chain terminating inhibitor of DNA synthesis by yeast pol eta, but not by the Klenow fragment. We attributed this selective inhibition to the unconstrained active site of pol eta compared to the Klenow fragment which is unable to hold on tightly to the templating nucleotide.  Because of this pyrene nucleotide is able to displace the templating nucleotide and bind tightly to the active site because of its superior pi-stacking ability.  Insertion of pyrene nucleotide is sensitive to sequence and is preferentially inserted opposite and following pyrimidines.  While the pi stacking energy of pyrene nucleotide on the 5'-end of DNA in a single sequence context has been previously determined, nothing is known about its stacking ability on the 3'-end of DNA which corresponds to a primer terminus.  We therefore determined the effect of a 3'-dangling pyrene on the thermodynamics of DNA melting as a function of the flanking base pair.  We synthesized oligodeoxynucleotides d(YGCGCGCXP) where X is A, C, G or T and Y is the complementary base by automated phosphoramidite synthesis via CPG-supported 5'-DMT pyrene nucleotide.  The stability of the duplexes paralleled the observed sequence effects on pyrene nucleotide insertion by pol h.  Most recently, we have extended our studies to 5-nitroindole nucleotide which has an almost identical free energy of stacking to that of pyrene nucleotide on the 5'-end of DNA.  Preliminary studies show that in spite of a similar pi-stacking energy, 5-nitroindole triphosphate is a less potent inhibitor for pol eta synthesis suggesting that factors other than stacking ability are also important.