Fig 1.
Structure of the guanine riboswitch aptamer domain and SK4 analog.
(A) Cartoon representation of the aptamer domain of the xpt riboswitch in complex with SK4 (colored spheres). Riboswitch regions are color-coded as follows: P1 (blue), P2, P3 (orange), L1, L2 (red), J1-2 (yellow), J2-3 (violet) and J3-1 (green) where P = paired, L = loop, J = junction (B) Chemical structure of SK4 showing modifications at the C2 (red) and C6 (blue) positions. Figures were created using VMD software (A) and ChemDraw Professional 23.0 (B).
Fig 2.
Analog SK4 was prepared in a four-step protocol starting from commercially available guanosine. The synthesis required acetyl protection (SK1) of the ribose hydroxyl groups and chlorination at C6 (SK2) before the desired modifications were made at C2 and C6 (SK3 and SK4).
Fig 3.
Miller assay results in the presence of ligand analogs.
Beta galactosidase reporter gene assay showing the expression of guanine riboswitch in the presence of DMSO control (red), guanine (orange), guanosine (yellow), and SK4 (green). Samples were all different from the DMSO control and were tested at three different concentrations. Significance differences were estimated using an analysis of variance followed by a post-hoc Tukey test (i.e., P < 0.0001, ***, P > 0.5, n.s.). Box plots show the median as well as the interquartile range. Individual points represent normalized miller units for each trial.
Fig 4.
RT qPCR riboswitch expression in the presence of various ligands.
Quantitative PCR was used to determine expression of riboswitch RNA in B. subtilis cells at 50 μM guanine (orange), guanosine (yellow) and SK4 (green). GAPDH mRNA expression was utilized as an internal control for all three samples where GN is guanine and GNS is guanosine. All samples were run in quadruplicates and were different from their respective controls (red). Crucially, SK4 was also statistically different from Guanosine and Guanine. Significance differences were estimated using an analysis of variance followed by a post-hoc Tukey test (i.e., P < 0.0001, ***, P > 0.5, n.s.).
Fig 5.
Molecular Dynamics simulation of riboswitch aptamer domain.
(A) Root-mean square deviations (RMSD) over 1000 ns, and (B) region-wise root-mean square fluctuations (RMSF) of the aptamer domain. Six different simulations were carried out. Apo crystal 6UBU with native ligand removed (black), crystal with native guanine (red), crystal with docked guanine (green), crystal with docked guanosine (blue), crystal with docked SK4 (purple), ensemble structure with docked SK4 (cyan).
Table 1.
Hydrogen bonding data from molecular dynamics simulations.