TEMPO-derived spin labels linked to the nucleobases adenine and cytosine for probing local structural perturbations in DNA by EPR spectroscopy

Abstract

Three 2'-deoxynucleosides containing semi-flexible spin labels, namely T A, U A and U C, were prepared and incorporated into deoxyoligonucleotides using the phosphoramidite method. All three nucleosides contain 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) connected to the exocyclic amino group; T A directly and U A as well as UC through a urea linkage. T A and U C showed a minor destabilization of a DNA duplex, as registered by a small decrease in the melting temperature, while U A destabilized the duplex by more than 10 °C. Circular dichroism (CD) measurements indicated that all three labels were accommodated in B-DNA duplex. The mobility of the spin label T A varied with different base-pairing partners in duplex DNA, with the T A ·T pair being the least mobile. Furthermore, T A showed decreased mobility under acidic conditions for the sequences xs T A·C and T A·G, to the extent that the EPR spectrum of the latter became nearly superimposable to that of T A ·T. The reduced mobility of the T A·C and T A·G mismatches at pH 5 is consistent with the formation of T AH + ·C and TAH + ·G, in which protonation of N1 of A allows the formation of an additional hydrogen bond to N3 of C and N7 of G, respectively, with G in a syn-conformation. The urea-based spin labels UA and UC were more mobile than T A, but still showed a minor variation in their EPR spectra when paired with A, G, C or T in a DNA duplex. U A and U C had similar mobility order for the different base pairs, with the lowest mobility when paired with C and the highest when paired with T.