The thermal stability of a G-quartet formed by incorporation of 8-methylguanine (m8G) in the DNA sequence at the 5' terminus of the Rb gene was examined by CD melting experiments. Substitutions of m8G at positions that show syn conformations in antiparallel G-quartets stabilize the structure. Formation of the G-quartet structure in the Rb gene acts as a barrier in DNA synthesis, as determined using a rapid and simple polymerase arrest assay. It was found that the C-rich complementary strand could form an i-motif structure at lower pH values.