Electrospray ionization mass spectrometry (ESI-MS) combined with fluorescence, circular dichroism, UV spectrophotometer, and native polyacrylamide gel electrophoresis techniques are used to study structural features of interlocked dimers formed by DNA sequence 93del (GGGGTGGGAGGAGGGT) and its derivatives. Herein, we demonstrate that the interlocked dimers can be distinguished from stacked dimers formed by sequences T30923 (GGGTGGGTGGGTGGGT) and T30177 (GTGGTGGGTGGGTGGGT). In addition, loop length, the base at 5'-end, and the isolation of T and TT to the first 4G tract do significantly influence the formation and topologies of interlocked dimers. Furthermore, our results suggest that the 4G tract and the 2G tract in various locations in the 93del derivative sequence can form interlocked structure. This work not only provides new insight into the assembly of 3 + 1 interlocked DNA conformations but also demonstrates that ESI-MS combined with other analytical methods is rapid and useful for DNA structural studies.