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A·C mismatches are studied by Raman spectral characterization of PolyA, PolyC, and their equimolar complex in solution of 0.14 mol/L Na , pH7.0. Experimental results show that A·C mismatches occur to be A/B (mainly A) conformers, and unlike Watson-Crick base pairing, this kind of mismatches is stabilized by only one hydrogen bond involving cytosine N4H2 and adenine N7. The formation of A·C complex makes the base stacking interactions much stronger, and conformation of the backbone more ordered, which leads to obvious Raman hypochromic effect with some shifts in corresponding bands.