Noncovalent and site-directed spin labeling of nucleic acids.

Electron paramagnetic resonance (EPR) spectroscopy is widely used to study free radicals or paramagnetic centers associated with biopolymers. With the advent of pulsed EPR methods, which allow accurate distance measurements between 20 and 80 , structures of biopolymers have increasingly been interrogated by this technique. Some of the advantages of EPR spectroscopy over other structural techniques are its sensitivity, that it is not restricted by molecular size, and that measurements can be performed under biological conditions. However, stable free radicals, such as nitroxide spin labels, must be incorporated into the biopolymers prior to EPR studies. In site-directed spin labeling (SDSL), spin labels are covalently attached to the biopolymers at a specific site of interest. For nucleic acids there have been two main strategies for SDSL. First, spin labels have been incorporated during automated oligonucleotide synthesis by employing spin-labeled phosphoramidite building blocks. This approach has the advantage that very sophisticated and structurally complex labels can be incorporated at specific sites. However, the synthetic challenges of spin-labeled phosphoramidites can be considerable. Furthermore, spin labels can be partially reduced upon exposure to the reagents used in the automated synthesis of oligonucleotides. The second SDSL approach is post-synthetic modification of the biopolymer. Here, a spin-labeling reagent is incubated with an oligonucleotide that contains a reactive functional group at a specific site. Post-synthetic labeling is in general less labor intensive than the phosphoramidite strategy, but drawbacks include incomplete labeling and side reactions of the spin label with inherent functional groups of the nucleic acids, such as the exocyclic amino groups of the nucleobases. Both strategies usually require purification of the spin-labeled material, which can be nontrivial. Here we report a new and straightforward SDSL protocol for nucleic acids that is based on noncovalent labeling. The new approach utilizes a nitroxide that is structurally related to the rigid spin label Ç. The spin label Ç is an analogue of cytidine (C), with a nitroxide-bearing isoindol moiety fused to cytosine by an oxazine linkage, and forms a stable Watson–Crick base pair with guanine (Figure 1). The rigidity of Ç enabled precise distance measurements by EPR, determination of the relative angular orientation between two spin labels, and has been used to study DNA dynamics and folding. 7] The strategy for noncovalent labeling was to disconnect the glycosidic bond of Ç to give an abasic site (F) and the free spin-labeled base (Figure 1). The spin label would bind in the abasic site through receptor–ligand interactions involving hydrogen bonding and p-stacking interactions. The synthesis of spin label started with regioselective alkylation of 5-bromouracil at N1 by a one-pot, two-step procedure using HMDS and benzyl bromide in the presence of a catalytic amount of iodine to obtain compound 2 (Scheme 1). Activation of 2 by conversion to the Osulfonylated derivative, followed by coupling with isoindol amino phenol derivative 4 yielded conjugate 5. Subsequent ring closure, facilitated by cesium fluoride, yielded phenoxazine derivative 6. Removal of the N1-protecting benzyl group by boron tribromide and oxidation of the amine to a nitroxide with mCPBA gave spin label . The EPR spectrum of in an aqueous solution containing ethylene glycol (30%) shows three narrow lines that broaden on reducing the temperature from 0 to 30 8C (Figure 2, left), due to slower tumbling of in solution. On mixing a DNA duplex containing an abasic site with , a slow-moving component appears in the EPR spectrum (shown by arrows, Figure 2 middle), indicating binding of the spin label to the abasic site. On further cooling, the extent of spin-label binding increased, and at 30 8C the narrow lines (the fastmotion component of the spectrum) had completely disappeared, consistent with the spin label being fully bound. For comparison, EPR spectra of a covalently Ç-labeled 14-mer were recorded under identical conditions (Figure 2, right). The mobility of the spin label that is covalently linked to the dsDNA is the same as that of the slow-moving component in the sample containing and the abasic DNA (Figure 2, Figure 1. a) Base-pairing scheme of spin labels Ç and with G. dR is 2’-deoxyribose. b) Structure of an abasic site in DNA.