A Novel Paramagnetic Relaxation Enhancement Tag for
Nucleic Acids: A Tool to Study Structure and Dynamics of RNA
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Abstract
In this work, we present a novel
2,2,6,6-tetramethylpiperidine
1-oxyl (TEMPO) radical phosphoramidite building block, which can be
attached to the 5′-terminus of nucleic acids. To investigate
the paramagnetic relaxation enhancement (PRE) emanating from this
radical center, we incorporated the TEMPO label into various types
of RNAs. We measured proton PREs for selectively <sup>13</sup>C-isotope
labeled nucleotides to derive long-range distance restraints in a
short 15 nucleotide stem–loop model system, underscoring the
potential of the 5′-TEMPO tag to determine long-range distance
restraints for solution structure determination. We subsequently applied
the distance-dependent relaxation enhancement induced by the nitroxide
radical to discern two folding states in a bistable RNA. Finally,
we investigated the fast conformational sampling of the HIV-1 TAR
RNA, a paradigm for structural flexibility in nucleic acids. With
PRE NMR in combination with molecular dynamics simulations, the structural
plasticity of this RNA was analyzed in the absence and presence of
the ligand l-argininamide