Semisynthetic alphabet can potentially increase the genetic information stored in DNA through the formation of unusual base pairs such as dSSICS:dNaM. However, recent experiments show that near-visible-light irradiation on the d5SICS and dNaM chromophores could lead to genetic mutations and damages. Until now, their photophysical mechanisms remain elusive. Herein, we have employed MS-CASPT2//CASSCF and QM(MS-CASPT2//CASSCF)/MM methods to explore the spectroscopic properties and excited state relaxation mechanisms of d5SICS, dNaM, and dSSICS:dNaM in DNA. We have found that (1) the S-2 state of d5SICS, the S-1 state of dNaM, and the S-2 state of dSSICS:dNaM are initially populated upon near-visible-light irradiation and (2) for d5SICS and dSSICS:dNaM, there are several parallel relaxation pathways to populate the lowest triplet state, but for dNaM, a main relaxation pathway is uncovered. Moreover, we have found that the excited-state relaxation mechanism of dSSICS:dNaM in DNA is similar to that of the isolated d5SICS chromophore. These mechanistic insights contribute to the understanding of photophysics and photochemistry of unusual base pairs and to the design of better semisynthetic genetic alphabet.