Effect of Molecular Crowding on the Stability of RNA G-Quadruplexes with Various Numbers of Quartets and Lengths of Loops.

G-Quadruplexes are noncanonical structures formed by guanine-rich regions of not only DNA but also RNA. RNA G-quadruplexes are widely present in the transcriptome as mRNAs and noncoding RNAs and take part in various essential functions in cells. Furthermore, stable RNA G-quadruplexes control the extent of biological functions, such as mRNA translation and antigen presentation. To understand and regulate the functions controlled by RNA G-quadruplexes in cellular environments, which are molecularly crowded, we would be required to investigate the stability of G-quadruplexes in molecular crowding. Here, we systematically investigated the thermodynamic stability of RNA G-quadruplexes with different numbers of G-quartets and lengths of loops. The molecular crowding conditions of polyethylene glycol with an average molecular weight of 200 (PEG200) were found to stabilize RNA G-quadruplexes with three and four G-quartets, while G-quadruplexes with two G-quartets did not exhibit any stabilization upon addition of PEG200. On the other hand, no difference in stabilization by PEG200 was observed among the G-quadruplexes with different loop lengths. Thermodynamic analysis of the RNA G-quadruplexes revealed more appropriate motifs for identifying G-quadruplex-forming sequences. The informatics analysis with new motifs demonstrated that the distributions of G-quadruplexes in human noncoding RNAs differed depending on the number of G-quartets. Therefore, RNA G-quadruplexes with different numbers of G-quartets may play different roles in response to environmental changes in cells.