A nucleotide-independent cyclic nitroxide label for monitoring segmental motions in nucleic acids.

Phuong H Nguyen
Phuong H Nguyen
Institute of Physical and Theoretical Chemistry
Anna M Popova
Anna M Popova
University of Southern California
Kalman Hideg
Kalman Hideg
University of Pécs
Peter Z Qin
Peter Z Qin
University of Southern California
United States

BMC Biophys 2015 9;8. Epub 2015 Apr 9.

Department of Chemistry, University of Southern California, 840 Downey Way, Los Angeles, CA 90089-0744 USA.

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.
PDF Download - Full Text Link
( Please be advised that this article is hosted on an external website not affiliated with PubFacts.com)
Source Status
http://dx.doi.org/10.1186/s13628-015-0019-5DOI ListingPossible
April 2015
6 Reads

Similar Publications

Site-specific DNA structural and dynamic features revealed by nucleotide-independent nitroxide probes.

Biochemistry 2009 Sep;48(36):8540-50

Department of Chemistry, University of Southern California, Los Angeles, California, USA.

In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Read More

View Article
September 2009

Motions of the substrate recognition duplex in a group I intron assessed by site-directed spin labeling.

J Am Chem Soc 2009 Mar;131(9):3136-7

Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744, USA.

The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. Read More

View Article
March 2009

A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids.

Nucleic Acids Res 2007 21;35(10):e77. Epub 2007 May 21.

Department of Chemistry, University of Southern California, LJS-251, 840 Downey Way, Los Angeles, CA 90089-0744, USA.

In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 5' terminus of nucleic acids of arbitrary sizes. Read More

View Article
June 2007

Site-directed spin-labeling of nucleic acids by click chemistry: detection of abasic sites in duplex DNA by EPR spectroscopy.

J Am Chem Soc 2010 Aug;132(30):10424-8

Department of Physics and Chemistry, University of Southern Denmark, Nucleic Acid Center Campusvej 55, 5230 Odense, Denmark.

This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis and purification of the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route, was reacted with an oligomer containing 5-ethynyl-2'-dU. Read More

View Article
August 2010