Abstract
BackgroundSpin 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.ResultsWe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-015-0019-5) contains supplementary material, which is available to authorized users.
Highlights
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
A post-synthesis scheme for incorporating a cyclic nitroxide label in nucleic acids To incorporate the cyclic R5c, the bi-functionalized R5c precursor was reacted with a nucleic acid strand containing ps modifications introduced at two consecutive nucleotides during solid-phase chemical synthesis (Figure 1A)
Data reported here clearly demonstrate that the cyclic R5c nitroxide can be efficiently attached to a given nucleic acid post-synthetically and under mild biochemical conditions
Summary
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. Spin labels refer to chemically stable radicals attached at macromolecules, and are utilized to study structure and dynamics of bio-molecules in techniques such as sitedirected spin labeling (SDSL) and paramagnetic NMR. Many SDSL studies use pyrroline- or piperidinebased nitroxides that are covalently attached at a specific site of the target macromolecule. These labels can be categorized into two groups based on the nature of chemical coupling between the target molecule and the nitroxide moiety bearing the unpaired electron. Examples of flexible labels include the prototypic R1 label for proteins, where the pyrroline ring is connected via a disulfide bond to a cysteine [1]; and the R5 and R5a nitroxides, where the pyrroline ring is connected via single-bonds
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