Jeremy S. Lee

AC impedance spectroscopy of native DNA and M-DNA.

Monolayers of thiol-labeled DNA duplexes of 15, 20, and 30 basepairs were assembled on gold electrodes. Electron transfer was investigated by electrochemical impedance spectroscopy with Fe(CN)(6)(3-/4-) as a redox probe. The spectra, in the form of Nyquist plots, were analyzed with a modified Randles circuit which included an additional component in parallel, R(x), for the resistance through the DNA. For native B-DNA R(x) and R(ct), the charge transfer resistance, both increase with increasing length. M-DNA was formed by the addition of Zn(2+) at pH 8.6 and gave rise to characteristic changes in the Nyquist plots which were not observed upon addition of Mg(2+) or at pH 7.0. R(x) and R(ct) also increased with increasing duplex length for M-DNA but both were significantly lower compared to B-DNA. Therefore, electron transfer via the metal DNA film is faster than that of the native DNA film and certain metal ions can modulate the electrochemical properties of DNA monolayers. The results are consistent with an ion-assisted long-range polaron hopping mechanism for electron transfer.

Immunofluorescent staining of mammalian nuclei and chromosomes with a monoclonal antibody to triplex DNA.

Triplex DNA is an unusual conformation of DNA formed when two pyrimidine nucleotide strands share a common purine strand. A monoclonal antibody, demonstrated by numerous criteria to be specific for triplex DNA, was used to investigate the presence and distribution of this unique DNA configuration in nuclei and chromosomes of mouse LM cells and human lymphocytes. Indirect immunofluorescence microscopy revealed that constitutive heterochromatin in acetic-methanol fixed mouse nuclei was usually, but not always immunofluorescent, suggesting possible cell cycle related variations in the amount of triplex DNA or its accessibility in this condensed chromatin. In fixed mouse and human chromosomes, there was a positive correlation between immunofluorescent staining patterns, Hoechst 33258 banding, and G- and/or C-banding patterns. Unfixed, isolated mouse chromosomes also reacted positively with the antibody, particularly when they were gently decondensed by exposure to low ionic conditions at neutral pH. This result indicates that fixation is not mandatory for antibody staining, suggesting that some mammalian chromosomal DNA may be naturally organized in a triplex configuration. However, there is a possibility that fixation may facilitate the formation of additional triplex DNA complexes in potential sequences or expose previously inaccessible triplex DNA. The precise correspondence between the immunofluorescent patterns produced by anti-triplex DNA antibodies and G- and C-bands known to represent regions of chromatin condensation, suggests a potential role of triplex DNA in chromosome structure and regional chromatin condensation.

Thermodynamic investigation of M-DNA: a novel metal ion–DNA complex

The thermodynamics of formation of a novel divalent metal ion-DNA complex known as M-DNA have been investigated using an ethidium bromide (EB) fluorescence assay, and with isothermal titration calorimetry. The process of M-DNA formation was observed from the EB assay to be strongly temperature-dependent. The binding of Zn(2+) to calf thymus (42% GC content) and Escherichia coli (50% GC content) DNA at pH 8.5 exhibited an endothermic cooperative binding process at Zn(2+) concentrations of approximately 0.1 mM, indicating an entropy driven process. This binding process is consistent with a site-specific binding interaction, similar in nature to Z-DNA formation; however, the interaction occurs at much lower metal ion concentrations. The enthalpy of M-DNA formation for calf thymus DNA was determined to be 10.5+/-0.7 and 9+/-2 kJ/mbp at DNA concentrations of 100 and 50 microg ml(-1), respectively. An enthalpy of 13+/-3 kJ/mbp was obtained for M-DNA formation for 50 microg ml(-1) E. coli DNA. No evidence of M-DNA formation was observed in either DNA at pH 7.5 with Zn(2+) or at either pH 7.5 or 8.5 with Mg(2+).

Nanopore detection of antibody prion interactions

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an alpha-hemolysin pore embedded in a lipid membrane. A prion peptide, PrP(143-169), can readily translocate through the pore, but on the addition of monoclonal antibody M2188, which binds the peptide, the number of translocations is reduced because the complex is too large to translocate. At a peptide-to-immunoglobulin G (IgG) ratio of 2:1, only bumping events were observed. The event profile of a control peptide that does not bind the antibody was unchanged. Similarly, the presence of the antibody prevents translocation of the full-length prion protein. Because a nanopore can detect a single molecule, these experiments represent an important first step towards the development of a sensitive prion detector.

Nanopore Analysis of the Folding of Zinc Fingers

In classical biochemistry, denatured or unfolded proteins were considered to be non-functional or inactive. However, recent progress in proteomics and genomics research suggests that as many as 30% of eukaryotic proteins are unfolded or contain significant disordered regions. (These proteins are also known as natively unfolded and intrinsically disordered). Many such proteins become folded upon interaction with DNA, other proteins, or metal ions and have also been implicated in disease pathogenesis. Examples include p53 and cancer, prion proteins and mad-cow disease, and a-synuclein and Parkinson’s disease. Needless to say, disordered proteins have proven difficult to study by conventional techniques, such as X-ray crystallography or NMR, because multiple conformations coexist. Single-molecule techniques may be used to overcome this problem as, by necessity, they examine a single conformation. Here, we demonstrate a simple model for conformational studies of protein folding involving the interaction of metal ions with Zn-finger peptides investigated by nanopore analysis. Recently, there have been several reports on the use of nanopores to investigate short peptides and small proteins, as well as antibody/protein and DNA/ protein complexes. The biological pore formed by a-hemolysin has proved to be very useful for this work since it self-assembles into a lipid membrane, yielding a channel with an internal diameter of about 1.5 nm. Under a constant voltage of 100mV in a buffer of 1 M KCl there is a constant current of about 100 pA, which is readily measured by patchclamp techniques. When a molecule approaches or enters the pore it causes a blockade current (I) for a particular time (T). The value of these parameters can be related to the charge, structure, and state of complexation of the molecule. If a protein is larger than the size of the pore then it may bump into the pore and then diffuse away which, in general, is characterized by a small value for I. Alternatively, the protein may unfold and translocate the pore, giving rise to a large I. Thus this technique can be used to study the state of folding of proteins and peptides at the single-molecule level.

Triplex DNA in plasmids and chromosomes

Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure.

Divalent cations induce a compaction of intrinsically disordered myelin basic protein.

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.

Nanopore analysis of the interaction of metal ions with prion proteins and peptides.

Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106-126 (PrP106-126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an alpha-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106-126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

Immunofluorescent localization of triplex DNA in polytene chromosomes of Chironomus and Drosophila

Purine · pyrimidine (pur·pyr) DNA tracts are prevalent in eukaryotic genomes. They can adopt a triplex conformation in vitro under conditions that may exist in vivo, suggesting that triplex (H-) DNA may exist naturally in chromosomes. To explore this possibility and gain insight concerning potential functions, the distribution of triplex DNA was studied in fixed polytene chromosomes of Chironomus tentans and Drosophila melanogaster by indirect immunofluorescence microscopy using an anti-triplex DNA monoclonal antibody (Jel 318). Chromosomes stained with this antibody exhibited immunopositive regions corresponding to condensed chromatin bands; interbands were less immunofluorescent. These results imply that there is more triplex DNA in bands than in interbands. In Chironomus, nucleolar organizer regions and Balbiani rings were immunonegative, indicating that triplex DNA is not present in decondensed, transcriptionally active chromatin. A few specific bands in both Chironomus and Drosophila were intensely immunofluorescent. In Drosophila, one such region was 81F on chromosome 3R. Competition during staining with exogenously added sequences corresponding to a constituent 1.672 g/cm3 satellite DNA in region 81F failed to abolish the immunofluorescence, suggesting that the satellite DNA does not fortuitously react with Jel 318 and implying that unidentified pur·pyr sequences forming triplex DNA are also present at this location. Region 81F exhibits ectopic pairing with nonrelated chromosome regions that have also proven to be intensely immunopositive; this suggests that the formation of triplex DNA between common, shared pur·pyr sequences in these otherwise nonhomologous bands might account for the ectopic pairing phenomenon. Together with our previous results, these data are consistent with the hypothesis that triplex DNA may play a role in chromosome organization by participating in regional chromatin condensation.

Cloning and expression of an autoimmune DNA-binding single chain FV. only the heavy chain is required for binding

Hed 10 is a murine autoimmune antibody which binds tightly to the single-stranded DNA, poly(dT). The heavy and light chain variable region genes of Hed 10 were cloned and then joined by a 42 base-pair linker with the aid of a PCR ligation technique to produce a single chain Fv gene. After insertion into an expression vector the single chain Fv protein (scFv Hed 10) was produced in high yield and was purified by chromatography on an oligo (dT) cellulose column. The binding of scFv Hed 10 to poly (dT) was measured by fluorescence quenching and a binding constant of 3.2 x 10(6) M-1 was calculated. Previously [Lee et al. (1982) Biochemistry 21, 4940-4945] the binding constant of Fab Hed 10 to poly (dT) was found to be 12.7 x 10(6) M-1. In addition the Vh gene of Hed 10 was expressed independently as well as another scFv which contained the Vh region of Hed 10 linked to the light chain variable region of Jel 42, an antibody specific for Hpr protein of E. coli (scFv 10 H.42 L). Both of these proteins had binding constants for poly (dT) in the range of 6 x 10(6) M-1. Therefore, the light chain of Hed 10 contributes little to the binding of this autoimmune antibody to DNA.