Noncovalent Spin‐Labeling of DNA and RNA Triplexes

Abstract

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine‐derived label and triplex motifs containing a single‐nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…Recently, G ́was also used to label DNA and RNA triplexes containing a single abasic site. 414 The N-oxide phenazine 136 is the latest noncovalent spin label that can bind to an abasic site in DNA but is unsuitable for RNA. 415 If the spin label can directly bind to RNA or DNA, no chemical modifications of oligonucleotides (not even the introduction of an abasic site) are required.…”

Spin‐Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block

“…The fold shown in Figure 4 was derived from enzymatic secondary structure mapping in absence of neomycin. [67] In contrast, in-line probing indicates restricted conformational mobility of G( 9) and U (10), presumably by forming base pairs with U(21) and C (22). This type of conformation, coaxial stacking of two helices while C(6) to U( 8) are expelled as a bulge, was found previously by enzymatic probing and NMR spectroscopy in aminoglycoside complexes of RNA 25.…”

“…Strong cleavage occurs after C(6), U (7), U (8) and in the loop region from U(13) to U (18). Interestingly, the peaks from G(9) to U (10) and U(21) to C (22) are weak. The fold shown in Figure 4 was derived from enzymatic secondary structure mapping in absence of neomycin.…”

“…Nitroxide radicals [1–3] are among the most useful spin labels to study the structure and dynamics of RNA by EPR spectroscopy [4–38] . They can be covalently attached to the base, sugar, or the phosphates of nucleotides [39–42] .…”

“…Although long‐living under air and in neutral aqueous buffer, reaction conditions typical for the solid‐phase synthesis of oligonucleotides and for enzymatic ligation steps are known to cause partial decomposition of nitroxides. Accordingly, spin labels are introduced after chain assembly in many cases and even noncovalent attachment is often used [7,12,21–23] . The most direct way to incorporate spin labels into DNA and RNA, however, is the use of nitroxide modified nucleoside phosphoramidites.…”