title: Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs.
abstract: Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; Synthetic Biology Institute, University of California, Berkeley, Berkely, CA 94720, USA.
authors.forename: Scott F
authors.initials: SF
authors.lastname: Hickey
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Synthetic Biology Institute, University of California, Berkeley, Berkely, CA 94720, USA. Electronic address: mingch@berkeley.edu.
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D018360:Crystallography, X-Ray; D015195:Drug Design; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D058928:Riboswitch / Q000187:drug effects; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058928:Riboswitch; C085321:cyanine dye 5; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase