Rigid spin-labeled nucleoside Ç: a nonperturbing EPR probe of nucleic acid conformation

Abstract

Rigid spin-labeled nucleoside Ç, an analog of deoxycytidine that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by chemical synthesis. Thermal denaturation experiments and circular dichroism (CD) measurements showed that Ç has a negligible effect on DNA duplex stability and conformation. Nucleoside Ç was incorporated into several positions within single-stranded DNA oligomers that can adopt two hairpin conformations of similar energy, each of which contains a four-base loop. The relative mobility of nucleotides in the alternating C/G hairpin loops, 5′-d(GCGC) and 5′-d(CGCG), was determined by electron paramagnetic resonance (EPR) spectroscopy. The most mobile nucleotide in the loop is the second one from the 5′-end, followed by the third, first and fourth nucleotides, consistent with previous NMR studies of DNA hairpin loops of different sequences. The EPR hairpin data were also corroborated by fluorescence spectroscopy using oligomers containing reduced Ç (Çf), which is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained Ç at the end of the helix showed features that indicated dipolar coupling between two spins. These data are consistent with end-to-end duplex stacking in solution, which was only observed when G was paired to Ç, but not when Ç was paired with A, C or T.

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…885 The rigid spin-label cytosine analogue (40) has been synthesised and forms stable base pairs with guanine, and was used to study the motion within an oligonucleotides that can adopt two different hairpin conformations. 177 Raman spectroscopy has been used in a study with EM to investigate the global structure of DNA binding to the bacteriophage P22 terminase subunit. 847 Surface enhanced Raman spectroscopy (SERS) is a technique in which samples are adsorbed onto rough metal surfaces and relies upon the excitation of localised surface plasmons.…”

“…The analogue (40) forms a specific base pair with dG, and the mobility of it in oligonucleotides can be monitored by EPR. 177 In addition, (40) is highly fluorescent when the nitroxide is reduced (sodium sulphide) and the fluorescent signal intensity is dependent upon the nature of its complementary base, thus it is suggested that it is possible to identify the opposing base from the fluorescent signal. 178,179 The expanded cytosine analogue (41) has been used for triplex stabilisation, as the extended aromatic ring system projects into the major groove and aids stability of a third strand by additional stacking interactions.…”

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

“…Phosphoramidite 4 was used to synthesize several oligonulceotides for characterization of DNAs containing Ç [54]. Circular dicroism measurements showed characteristic spectra for B-form DNA.…”

Nitroxides and nucleic acids: Chemistry and electron paramagnetic resonance (EPR) spectroscopy

“…The fluorescent nucleoside is sensitive to its microenvironment and is able not only to detect mismatches, but also to identify the mismatched base [28,29]. The bifunctionality of the spectroscopic probe allows, for the first time, the use of the same nucleic acid sample for both EPR and fluorescence spectroscopies and has been applied for the study of the conformation and dynamics of hairpin loops [30,31] and the folding of a DNA aptamer that binds cocaine [32].…”