NMR GHZ

We describe the creation of a Greenberger-Horne-Zeilinger (GHZ) state of the form |000>+|111> (three maximally entangled quantum bits) using Nuclear Magnetic Resonance (NMR). We have successfully carried out the experiment using the proton and carbon spins of trichloroethylene, and confirmed the result using state tomography. We have thus extended the space of entangled quantum states explored systematically to three quantum bits, an essential step for quantum computation.Comment: 4 pages in RevTex, 3 figures, the paper is also avalaible at http://qso.lanl.gov/qc

Computational and theoretical aspects of biomolecular structure and dynamics

This is the final report for a project that sought to evaluate and develop theoretical, and computational bases for designing, performing, and analyzing experimental studies in structural biology. Simulations of large biomolecular systems in solution, hydrophobic interactions, and quantum chemical calculations for large systems have been performed. We have developed a code that implements the Fast Multipole Algorithm (FMA) that scales linearly in the number of particles simulated in a large system. New methods have been developed for the analysis of multidimensional NMR data in order to obtain high resolution atomic structures. These methods have been applied to the study of DNA sequences in the human centromere, sequences linked to genetic diseases, and the dynamics and structure of myoglobin

Use of Larval Morphological Deformities in Chironomus plumosus (Chironomidae: Diptera) as an Indicator of Freshwater Environmental Contamination (Lake Trasimeno, Italy)

The mentum deformity incidence in Chironomus plumosus larvae to assess the environmental contamination level in Lake Trasimeno, Central Italy, was investigated. The survey lasted from May 2018 to August 2019. Fifty-one samplings were carried out: 34 in the littoral zone and 17 in the central zone. The deformity assessment was based on 737 and 2767 larval specimens of C. plumosus collected from the littoral and central zones, respectively. Comparison of the larval morphometric variables between normal and deformed specimens highlighted that the deformities did not cause alterations of the larval growth. The deformity incidence amounted to 7.22% in the whole Trasimeno's ecosystem, reaching 8.28% in the littoral zone and 6.94% in the central zone. Among the different seasonal cohorts, the spring cohort had overall the highest deformity value (11.41%). The deformity type assessment protocol highlighted that the most common deformity type was "round/filed teeth" (64%). The results of this 2018–2019 survey revealed a low deformity incidence, within the background range of relatively low-impacted freshwaters. Comparison with previous investigations (2000–2010) of the same habitat showed a clear decrease of the deformity incidence. This study further contributes to the evaluation of the mentum deformity in chironomids that represent an indicator endpoint of the anthropogenic contamination level in freshwaters

Role of conformational dynamics in sequence-specific autoantibody•ssDNA recognition

11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at 31 V H to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that R31 V H contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8•ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8•ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at 31 V H . © 2007 Wiley Periodicals, Inc. Biopolymers 85: 481–489, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55985/1/20692_ftp.pd

The PNA–DNA hybrid I-motif: implications for sugar–sugar contacts in i-motif tetramerization

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA–DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H(+)-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H(+)-C base pairs per heteroduplex. The imino protons are protected from D(2)O exchange suggesting intercalation of the heteroduplexes as seen in DNA(4) i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA–DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1′–H1′ NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar–sugar interactions as seen in DNA(4) i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar–sugar contacts in i-motif tetramerization

Highly fluorescent guanosine mimics for folding and energy transfer studies

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2′-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2′-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2′-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2′-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02–0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (ηt) are highly structure selective, with ηt(duplex) < ηt(single-strand) < ηt(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM

Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells

Guanine-rich DNA of a particular sequence adopts four-stranded structural forms known as G-quadruplex or G4 DNA. Though in vitro formation of G4 DNA is known for several years, in vivo presence of G4 DNA was only recently noted in eukaryote telomeres. Recent bioinformatics analyses showing prevalence of G4 DNA within promoters of human and related species seems to implicate G4 DNA in a genome-wide cis-regulatory role. Herein we demonstrate that G4 DNA may present regulatory sites on a genome-wide scale by showing widespread effect on gene expression in response to the established intracellular G4 DNA-binding ligands. This is particularly relevant to genes that harbor conserved potential G4 DNA (PG4 DNA) forming sequence across human, mouse and rat promoters of orthologous genes. Genes with conserved PG4 DNA in promoters show co-regulated expression in 79 human and 61 mouse normal tissues (z-score > 3.5; P < 0.0001). Conservation of G4 DNA across related species also emphasizes the biological importance of G4 DNA and its role in transcriptional regulation of genes; shedding light on a relatively novel mechanism of regulation of gene expression in eukaryotes

Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression

Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif

Intramolecular Folding in Human ILPR Fragment with Three C-Rich Repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures