Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

Abstract

DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in cancer cells. They are also predicted to exist in the guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA), which are transcribed in the cell's nucleolus. Recent studies strongly suggest that these sequences are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis, but the structures of ribosomal G-quadruplexes at atomic resolution are unknown and very little biophysical characterization has been performed on them. In the present study, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA, reminiscent of the analogous telomeric quadruplex structures. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K+ or Na+ ions. Our preliminary MD simulations of the telomeric parallel G-quadruplex (TEL 24P) showed that the K+ ion is expelled within a few nanoseconds of simulations. We optimized the CHARMM36 force field K+ parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K+ ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. The antiparallel topology was determined to have a disordered configuration due to the lack of planarity after the simulation. Our study suggests that NUC 19 and NUC 23 form well-defined, predominantly parallel-topology G-quadruplexes that are potential nucleolar targets for the design of novel chemotherapeutics.