Rapid purification of histidine-tagged glutathione S-transferase fusion protein by metal chelate POROS perfusion chromatography media.

The use of POROS MC media allowed high speed column preparation, sample loading, and elution. Under these conditions the proteins of interest can be recovered under mild conditions to maintain maximum biological activity. The speed of the process also assures minimum degradation or post-extraction modifications of the protein of interest from exposure to naturally occurring proteases and other enzymes. It is evident from the analysis by polyacrylamide gel electrophoresis of the purified fusion protein and starting material that a high degree of purification is possible in a single high-speed separation on POROS MC. The high flow rate capabilities of POROS media offer several other significant advantages over conventional media. A few of these include high speed and systematic method optimization, rapid column preparation and sample loading, and fast time-saving chromatographic separations.

Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance.

Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.

Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization.

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.

Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues.

Conformation detection and base dynamics of spin-labeled Z-DNA have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The two synthesized and characterized probes used in this study were C(5)-nitroxide-labeled 2'-deoxycytidine 5'-triphosphates, pppDCAT and pppDCAVAT, which serve as suitable substrates for Micrococcus luteus DNA polymerase. Enzymatic incorporation of these probes into (dG-dC)n yields the EPR-active alternating copolymers (dG-dC,DCAT)n and (dG-dC,-DCAVAT)n. These polymers assume typical B- and Z-DNA conformations under respective low (0.1 M NaCl) and high (4.5 M NaCl) salt conditions, as evidenced by their UV-circular dichroism spectra. The EPR line shape of (dG-dC,DCAT)n in Z-form is unique and significantly different from the B-form EPR spectrum. A similar observation is made for (dG-dC,DCAVAT)n. Thus, the EPR line shapes of these spin-labeled DNAs are indicative of their local conformations. The EPR spectra, analyzed with a previously published motional model [Kao, S.-C., Polnaszek, C.F., Toppin, C.R., & Bobst, A.M. (1983) Biochemistry 22, 5563-5568], indicate tau perpendicular values of 4 and 7 ns for the B- and Z-forms, respectively. Therefore, the base dynamics of Z-DNA are about two times slower than in B-DNA.

An electron paramagnetic resonance probe to detect local Z-DNA conformations.

We present spectroscopic evidence for an electron paramagnetic resonance (EPR) probe to detect local Z-DNA conformations in synthetic DNA. A spin labeled deoxycytidine-5'-triphosphate (pppDCAT) was co-polymerized with Micrococcus luteus DNA polymerase to yield the spin active alternating co-polymer (dG-dC,DCAT)n. The EPR spectrum of (dG-dC,DCAT)n in the Z-DNA conformation indicates a decrease in the local base dynamics by about a factor of two as compared to that computed for B-DNA. A control experiment conducted with (dA-dT, DUAT)n under similar salt conditions rules out the possibility of observing salt induced artifacts.

Nick translation of lambda phage DNA with a deoxycytidine analog spin labeled in the 5 position.

The synthesis and properties of a novel C(5)-spin-labeled 2'-deoxycytidine 5'-triphosphate which serves as a suitable substrate for the template-directed enzyme Escherichia coli DNA polymerase I are reported. The spin label is readily incorporated into lambda phage DNA by nick translation where it reports the characteristic local base motion for double- and single-stranded DNA as determined by electron spin resonance. The high-frequency deoxycytidine motion is similar to the previously reported thymidine motion in double-stranded lambda phage DNA.