title: A nucleotide-independent cyclic nitroxide label for monitoring segmental motions in nucleic acids.
abstract: BACKGROUND
  Spin labels, which are chemically stable radicals attached at specific sites of a bio-molecule, enable investigations on structure and dynamics of proteins and nucleic acids using techniques such as site-directed spin labeling and paramagnetic NMR. Among spin labels developed, the class of rigid labels have limited or no independent motions between the radical bearing moiety and the target, and afford a number of advantages in measuring distances and monitoring local dynamics within the parent bio-molecule. However, a general method for attaching a rigid label to nucleic acids in a nucleotide-independent manner has not been reported.
  
  
  RESULTS
  We developed an approach for installing a nearly rigid nitroxide spin label, designated as R5c, at a specific site of the nucleic acid backbone in a nucleotide-independent manner. The method uses a post-synthesis approach to covalently attach the nitroxide moiety in a cyclic fashion to phosphorothioate groups introduced at two consecutive nucleotides of the target strand. R5c-labeled nucleic acids are capable of pairing with their respective complementary strands, and the cyclic nature of R5c attachment significantly reduced independence motions of the label with respect to the parent duplex, although it may cause distortion of the local environment at the site of labeling. R5c yields enhanced sensitivity to the collective motions of the duplex, as demonstrated by its capability to reveal changes in collective motions of the substrate recognition duplex of the 120-kDa Tetrahymena group I ribozyme, which elude detection by a flexible label.
  
  
  CONCLUSIONS
  The cyclic R5c nitroxide can be efficiently attached to a target nucleic acid site using a post-synthetic coupling approach conducted under mild biochemical conditions, and serves as a viable label for experimental investigation of segmental motions in nucleic acids, including large folded RNAs.
authors.affiliation: Department of Chemistry, University of Southern California, 840 Downey Way, Los Angeles, CA 90089-0744 USA ; Current Address: Bachem Americas, Torrance, CA 90505 USA.
authors.forename: Phuong H
authors.initials: PH
authors.lastname: Nguyen
authors.affiliation: Department of Chemistry, University of Southern California, 840 Downey Way, Los Angeles, CA 90089-0744 USA ; Current Address: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037 USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Popova
authors.affiliation: Institute of Organic and Medicinal Chemistry, University of Pécs, Szigetic Strasse 12, Pécs, Hungary.
authors.forename: Kálmán
authors.initials: K
authors.lastname: Hideg
authors.affiliation: Department of Chemistry, University of Southern California, 840 Downey Way, Los Angeles, CA 90089-0744 USA.
authors.forename: Peter Z
authors.initials: PZ
authors.lastname: Qin
keywords: Dynamics; EPR; Nucleic acids; Rigid label; Site-directed spin labeling