i-Motif is formed from two parallel duplexes by intercalating with each other in an antiparallel orientation and each duplex is held together-via-hemiprotonated cytosine+-cytosine base pairs.However,a cell is crowded with various biomolecules and little is known about the properties of i-Motif under molecular crowding conditions until now.In the present study,we used human telomeric DNA sequence,[C3TA23C3,as a model system to investigate such problem by circular dichroism(CD) and UV absorbance spectroscopy.Based on the CD spectra,we found that there were no changes about the structure of i-Motif in the presence of polyethylene glycol(PEG).CD melting results showed that the thermal stability of i-Motif was increased under molecular crowding conditions compare to that in dilute buffer,which was further demonstrated by UV-melting results.This work suggests that molecular crowding could not affect the structure of i-Motif,which may depend on pH,while could enhance the thermal stability of i-Motif.