MONITORING DNA DYNAMICS USING SPIN-LABELS WITH DIFFERENT INDEPENDENT MOBILITIES

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyur idine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the ind ependent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the d iacetylene tether, there is considerable independent motion of the pro be. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is d emonstrated here that, when coupled via the diacetylene tether, the ni troxide undergoes a rapid uniaxial rotation about the tether. A detail ed analysis of the EPR spectrum of duplex DNA in solution, spin-labele d using the diacetylene tether, demonstrates that the motion of the ni troxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tu mbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex D NA with the monoacetylene-tethered nitroxide.