biostudies-literatureHoeher JENational Institutes of HealthNIGMS NIH HHSNIH HHS419-433https://www.ebi.ac.uk/biostudies/studies/S-EPMC10950518biostudies-literatureUnknown100(2)Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ₁ riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ₁ . We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ₁ at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ₁ concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ₁ riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.Photochemistry and photobiologyProbing and perturbing riboswitch folding using a fluorescent base analogue.PMC10950518R00 GM120457Sande NEHoeher JEWidom JRfalseProbing and perturbing riboswitch folding using a fluorescent base analogue.Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ₁ riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ₁ . We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ₁ at all labeling positions tested, although modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ₁ concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ₁ riboswitch and that 2-AP substitution exerts complex and varied impacts on this riboswitch.2024-01-01T00:00:00Z2024 Mar-Apr2026-06-02T10:23:20.464Z2025-04-06T08:32:32.53ZS-EPMC109505183809828710.1111/php.13896