Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules.

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

Cleavage of DNA by the insulin-mimetic compound, NH4[VO(O2)2(phen)].

The kinetics and mechanism of cleavage of DNA by the insulin-mimetic peroxo-vanadate NH4[VO(O2)2(phen)], pV, are described. In the presence of low energy UV radiation or biologically common reducing agents, pV decomposes into the monomer, dimer, and tetramer of vanadate and an uncharacterized compound of V4+ as shown by 51V NMR, ESR, and absorption spectra. The rate of photodecomposition of pV is reduced in the presence of calf thymus DNA, indicating that a decomposition product of the peroxo-vanadate, that is important in the destruction pathway of the complex, is interacting with DNA. This species, probably a short-lived complex of V4+, may also be responsible for the observed catalytic decomposition of pV in the absence of DNA by ascorbate. If closed circular pBR322 DNA is present when the peroxo-vanadate is destroyed by either UV radiation or reducing agents, the polymer may have its sugar-phosphate backbone broken. Closed circular DNA (form I) is converted into nicked circular DNA (form II) and linear DNA (form III). The amounts of the various forms produced as a function of irradiation time and peroxo-vanadate concentration were fit to a kinetic model to derive rate constants for the conversions. The kinetic analysis shows that pV is a single-strand nicking agent which exhibits some base and/or sequence preference. Furthermore, the pH dependences of the rates for conversion of form I to form II and for conversion of form II to form III are different, indicating that the nature of the chemistry at the site of cleavage on DNA influences further cutting by activated pV. Reduced amounts of DNA breakage in the presence of various salts and metal binding ligands indicate that a short-lived reactive complex of V4+, not the V4+ species detected by ESR at long irradiation times, is important in the cleavage process. The susceptibility of pV to decomposition by biologically common reducing agents suggests that metabolites of the agent, and not the compound itself, are responsible for its insulin-mimetic effects.

New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping.

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.

Chemically and photochemically initiated DNA cleavage by an insulin-mimetic bisperoxovanadium complex.

Chemically and photochemically induced cleavage of DNA by the insulin-mimetic compound NH4[VO(O2)2-(1,10-phenanthroline)], bpV(phen), have been studied. 51V NMR and absorption indicate that photoirradiation with low energy UV light of aqueous solutions containing bpV(phen) leads to the conversion of the compound to simple vanadates. Photoillumination of the compound in the presence of supercoiled pBR322 DNA results in cutting of the plasmid to produce nicked circular and linear DNA. Quantitative analysis of agarose gel data shows that bpV(phen) is a single strand nicking agent exhibiting sequence and/or base specificity.

Binding of delta- and lambda-[Ru(phen)3]2+ to [d(CGCGATCGCG)]2 studied by NMR.

The interactions of the delta and lambda enantiomers of the chiral metal complex [Ru(phen)3]2+ (phen = 1,10-phenanthroline) with the oligonucleotide duplex [d(CGCGATCGCG)]2 have been studied with NMR and CD spectroscopy. From NOESY data it is shown that the interaction primarily takes place in the minor groove of the oligonucleotide which remains in a B-like conformation. The observed NOEs also provide evidence that the metal complexes preferentially bind to the central AT region. The observed AT specificity is more pronounced with the delta as compared to the lambda enantiomer, which interacts with a larger part of the oligonucleotide. Furthermore, the NOESY data show that neither of the enantiomers binds by classical intercalation. This is also supported by a comparison study of the analogue [Ru(phen)2DPPZ]2+ (DPPZ = dipyrido[3,2-a:2',3'c]phenazine) which intercalates in DNA. The NMR as well as the CD results show that the delta and lambea enantiomers of [Ru(phen)3]2+ bind in different modes to [d(CGCGATCGCG)]2. Comparison of CD spectra of the metal complex in the presence of [d(CGCGATCGCG)]2, poly(dAdT).poly-(dAdT), poly(dGdC).poly(dGdC), and calf thymus DNA suggests that these binding modes are independent of DNA sequence. The results are found to be compatible with binding of delta-[Ru(phen)3]2+ by insertion of two phenanthroline ligands into the minor groove, causing minor distortions of the DNA structure, whereas the lambda enantiomer binds in a mode that leaves the DNA structure unaffected.

On the use of chiral compounds for probing the DNA handedness: Z to B conversion in poly(dGm5dC) upon binding of Fe(phen)3(2+) and Ru(phen)3(2+).

In order to examine whether chiral metal complexes can be used to discriminate between right- and left-handed DNA conformational states we have studied the enantioselective interactions of Fe(phen)3(2+) and Ru(phen)3(2+) (phen = 1,10-phenanthroline) with poly(dGm5dC) under B- and Z-form conditions. With the inversion-labile Fe(phen)3(2+), enantioselectivity leads to shifts in the diastereomeric binding equilibria. This effect, known as the "Pfeiffer effect" (1-4), is monitored as slowly emerging circular dichroism of the solution, corresponding to a net excess of the favoured enantiomer. With Ru(phen)3(2+), which is stable to intramolecular inversion, the difference in DNA-binding strengths of the enantiomers results in an excess of the less favoured enantiomer in the bulk solution. This excess is detected in the dialysate of the DNA/metal complex solution. With both complexes we find that the delta-enantiomer is favoured when the polynucleotide adopts the B-form, as previously shown, but also when it initially adopts the Z-form conformational state. This observation, together with evidence from UV-circular dichroism and binding data, indicates that the binding of these metal complexes induces a Z- to B-form transition in Z-form poly(dGm5dC). Consequently, neither of the studied chiral DNA-binders can easily be used to discriminate the DNA handedness.