Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization

Abstract

Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.

Structure and dynamics of a DNA-based model system for the study of electron spin–spin interactions

“…Paramagnetic manganese can substitute for magnesium in some catalytic RNAs [14][15][16][17][18] and a wide variety of spin-labeled nucleotides have been developed. [5,6,[19][20][21][22][23][24] The interactions of paramagnetic lanthanide ions with nucleic acids, directly or indirectly, are also of interest. The substitution of Er(III) for Mg(II) inhibits Dicer, a specialized ribonuclease while the substitution of La(III) for Mg(II) improves the processivity of reverse transcriptase, a DNA polymerase.…”

Synthesis and Electron Paramagnetic Resonance Studies of Oligodeoxynucleotides Containing 2‐N‐tert‐Butylaminoxyl‐2′‐deoxyadenosines

“…Only smalld ifferences wereo bserved in the T m values of the four duplexes with differing counterbases opposite A* (Figure 2A). [7,15] In the A* residue, the unpaired electron of the aminoxyl radical interacts with electrons of adenine because of the directc onnectiono ft he aminoxyl moiety to the adenine system.T his electronic property,w hich cyclic aminoxyls do not have, and the unique structure of A* might possibly influencet he EPR spectral shape of ODNs containing A*.H owever,t he exact reason why h 0 is larger than h 1 in the EPR spectra of single-stranded 1 is currently unclear. In the EPR spectra of the duplexes, peak broadening and decreased peak heightw ere most evident for 1:com(C).T oc omparet he spectra clearly,t he heights of three peaks in each spectrum-that is, the low-field peak (h 1 ), the center peak (h 0 ), and the high-field peak (h À1 ), relative to h 1 of 1-were plotted for 1 and for the duplexes ( Figure 2B).…”

“…Hybridization of 1 with com(T) resulted in peak broadening and ad ecrease in peak height similar to those observed for ODNs containing TEMPO and PROXYL systems( Figure 1). [15] Hybridization also caused considerable change in the low-and the high-field peaks. At 10 8C, ODN motion and the mobility of A* in 1 were decreased, and peak broadening and reduction of the peak heights were observed for 1 and 1:com(T).H ybridization was successfully monitored again.…”

Theory for Spin−Lattice Relaxation of Spin Probes on Weakly Deformable DNA

“…Hence, measurements in such systems provide information on the average behavior of the duplex rather than site-specific data. In order to examine properties of duplex DNA as a function of sequence and position, great effort has been expended in developing probes that can be covalently bound to specific sites for use in EPR 10,[17][18][19][20][21][22][23] and in labeling specific atoms by isotopic substitution to prepare sequences for NMR studies. 10,[24][25][26][27] EPR labeling experiments have focused on replacing natural bases with analogs, modified to contain the EPR active nitroxide radical as an integral part of the base.…”