Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs

Scott F Hickey; Ming C Hammond

doi:10.1016/j.chembiol.2014.01.004

Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs

Chem Biol. 2014 Mar 20;21(3):345-56. doi: 10.1016/j.chembiol.2014.01.004. Epub 2014 Feb 20.

Authors

Scott F Hickey¹, Ming C Hammond²

Affiliations

¹ Department of Chemistry, University of California, Berkeley, CA 94720, USA; Synthetic Biology Institute, University of California, Berkeley, Berkely, CA 94720, USA.
² 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.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites
Carbocyanines / chemistry
Crystallography, X-Ray
Drug Design*
Fluorescence Polarization
Fluorescent Dyes / chemistry*
High-Throughput Screening Assays
Kinetics
Methionine Adenosyltransferase / genetics
Methionine Adenosyltransferase / metabolism
Molecular Dynamics Simulation
Nucleic Acid Conformation
RNA, Bacterial / chemistry
RNA, Bacterial / metabolism
RNA, Messenger / chemistry*
RNA, Messenger / metabolism
Riboswitch* / drug effects
S-Adenosylmethionine / analogs & derivatives*
S-Adenosylmethionine / chemical synthesis
S-Adenosylmethionine / pharmacology
Staphylococcus aureus / enzymology

Substances

Bacterial Proteins
Carbocyanines
Fluorescent Dyes
RNA, Bacterial
RNA, Messenger
Riboswitch
cyanine dye 5
S-Adenosylmethionine
Methionine Adenosyltransferase

Abstract

Publication types

MeSH terms

Substances

Grants and funding