Formation of i-motif structure at neutral and slightly alkaline pH

It is well known that oligonucleotides containing tracts cytosines can form i-motif structures under acidic conditions (pH < 7). However, whether i-motif can be formed under normal physiological cellular conditions (pH 7.0-7.5) is yet no conclusive proof. In the present work, using circular dichroism (CD), UV absorption spectroscopies and native polyacrylamide gel electrophoresis (PAGE), we provided the compelling evidence for the formation of i-motif structures by four cytosine clusters, [C(3)TA(2)](3)C(3) (HT), [C(4)G](3)C(4)TA (RET), C(2)T(3)C(2)T(4)C(2)T(3)C(2) (CTC) and GC(2)GC(3)A(4)C(6)G (Rb), at neutral and slightly alkaline pH at 4 degrees C. Furthermore, for HT, we also supplied the evidence for the formation of i-motif structure by fluorescence resonance energy transfer (FRET) and investigated its folding kinetics. The formation time constants obtained by CD and fluorescence experiments are 214 and 493 s, respectively, indicating that HT can slowly form i-motif structure at pH 7.0 and 4 degrees C. This work implies that i-motif structures may possible form in vivo.