Bisulfite-free and single-nucleotide resolution sequencing of DNA epigenetic modification of 5-hydroxymethylcytosine using engineered deaminase

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed as a critical epigenetic modification. Deciphering the functions of 5hmC necessitates the location analysis of 5hmC in genomes. Here, we proposed an engineered deaminase-mediated sequencing (EDM-seq) method for the quantitative detection of 5hmC in DNA at single-nucleotide resolution. This method capitalizes on the engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine, 5mC, and 5hmC. In EDM-seq, the engineered A3A (eA3A) protein can deaminate C and 5mC but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A to form U and T, both of which are read as T during sequencing, while 5hmC is resistant to deamination by eA3A and is still read as C during sequencing. Therefore, the remaining C in the sequence manifests the original 5hmC. By EDM-seq, we achieved the quantitative detection of 5hmC in genomic DNA of lung cancer tissue. The EDM-seq method is bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the straightforward and quantitative detection of 5hmC in DNA at single-nucleotide resolution.

In EDM-seq, the original C and 5mC in DNA are deaminated by eA3A to form U and T, both of which are read as T during sequencing. While the 5hmC is resistant to deamination by eA3A and is still read as C during sequencing.     

Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein

We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu(2)O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein TraI in a room-temperature precursor electrolyte. The functional TraI derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu(2)O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu(2)O nanoparticles form, even though the concentrations of [Cu(+)] and [OH(-)] are at 5% of the solubility product (K(sp,Cu2O)). Negative control experiments verify that Cu(2)O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu(2)O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu(2)O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu(2)O (K(d) = 1.2 x 10(-)(8) M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.     

Spectral identification of specific photophysics of cy5 by means of ensemble and single molecule measurements

The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g., as a probe or fluorescence resonance energy transfer acceptor in large biomolecules. However, the unknown spectral properties of the triplet state and the lack of knowledge on the photoisomerization do not allow us to interpret precisely the unexpected single molecule behaviors. This limits the application of Cy5. The laser photolysis experiments demonstrate that the trans triplet state of Cy5 absorbs about 625 nm, the cis ground state absorbs about 690 nm, and the cis triplet state also absorbs about 690 nm. In other words, the T1-Tn absorptions largely overlap the ground-state absorptions for both trans and cis isomers, respectively. Furthermore, the observation of the cis triplet state indicates an important isomerization pathway from the trans-S1 state to the cis-T1 state upon excitation. The detailed spectra presented in this article let us clearly interpret the exact mechanisms responsible for several important and unexpected photophysical behaviors of single Cy5 molecules such as reverse intersystem crossing (RISC), the observation of dim states with a lower emission intensity and slightly red-shifted fluorescence, and unusual energy transfer from donor molecules to dark Cy5 molecules acting as acceptors in single molecule fluorescence resonance energy transfer (FRET) measurements. Spectral results show that the dim state in the single molecule fluorescence intensity time traces originated from cis-Cy5 because of a lower excitation rate, resulting from the red-shifted ground-state absorption of cis-Cy5 compared to that of the trans-Cy5.     

Toward the molecular flashlight: preparation,properties, and photophysics of a hypericin-luciferin tethered molecule

The synthesis of a molecule containing hypericin and luciferin moieties joined by a tether is reported. The light-induced (in vitro) antiviral activity as well as the photophysical properties of this new compound are measured and compared with those of the parent compounds, hypericin and pseudohypericin. This tethered molecule exhibits excited-state behavior that is very similar to that of its parent compounds and antiviral activity that is identical, within experimental error, to that of its more closely related parent compound, pseudohypericin. The implications for a photodynamic therapy that is independent of external light sources are discussed.     

Synthesis and conformational analysis of fructose-derived scaffolds: molecular diversity from a single molecule

Bi- and tricyclic compounds were synthesized starting from fructose. The different hydroxyl groups present in fructose were exploited in the formation of a number of conformationally constrained sugar-based scaffolds, including azido acids. Introduction of an azido group and carboxy terminus into different bicyclic iodo ethers, allowed the synthesis of different conformationally constrained azido acids. Conformational analysis of compounds 10, 11, 17, and 20 by NMR experiments assisted by molecular mechanics, allowed the determination of the distances between the relevant functional groups, that is the azido and carboxy functionalities.     

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2′-deoxyribonucleoside 5′-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.     

Detection and separation of nucleoside-5'-monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser-induced fluorescence detection

We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.     

Analysis of peptides by means of a single focusing mass spectrometer equipped with a molecular beam solid analysis/fast atom bombardment source

The use of a molecular beam solid analysis/fast atom bombardment source in conjunction with a single focusing mass spectrometer of limited specification is described for the identification of peptides. This ionization method is shown to have significant advantages for such analyses and the performance of the mass spectrometer is shown to be adequate for this work.     

Enhanced fluorescence of Cy5-labeled oligonucleotides near silver island films: a distance effect study using single molecule spectroscopy

We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.     

Self-assembly of a heterometallic molecular triangle using an ambidentate ligand and self-selection for a single linkage isomer

Self-assembly and self-selection of a single linkage isomer of a mixed-metal molecular triangle with ferrocene functionality have been achieved using an ambidentate ligand, and the product was characterized by multinuclear NMR spectroscopy and X-ray single crystal structure determination.     

Direct observation of triplet state emission of single molecules: single molecule phosphorescence quenching of metalloporphyrin and organometallic complexes by molecular oxygen and their quenching rate distributions

Single molecules are detected through the phosphorescence emission of their triplet states. Emission of the triplet states of single molecules of Pt octabutoxycarbonyl porphyrin (PtOBCP) and ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline dichloride (Ru(dpp)(3)Cl(2)) is reported. The single molecule phosphorescence is very sensitive to molecular oxygen. Each molecule has its own characteristic quenching rate by oxygen, and the distribution of these rates is measured for (Ru(dpp)(3)Cl(2)) on a quartz surface. The large variance of this distribution is presumed to be caused by fluctuations in the pseudobimolecular rate coefficient and the local oxygen concentration. The possibility of creating a quantitative single oxygen molecule sensor is suggested.     

Evaluation of effect of functionalized gold nanoparticles with a partial negative charge on stability of DNA molecule: a study of molecular dynamics simulation

Today, understanding the interaction between DNA molecule with nanoparticles and functionalized nanoparticles has a significant importance in medical applications and targeted drug delivery. Molecular dynamics simulation on double-stranded molecule with the structure of the double helix and sequence of (CCTCAGGCCTCC) was performed in three states. The aim was to evaluate the effect of gold nanoparticles (GNPs) with partial negative charge on the stability of a DNA molecule. During the simulation process, the GNPs become closed to the DNA molecule and phosphate groups of the DNA molecule guided the nanoparticles toward its major groove. At the end of the DNA molecule chain, the terminal nucleotide of the chain was laid flat on the surface of the GNPs due to excessive exposure to solvent molecules and occurrence of peeling and untwisting states. According to the results, proximity of the GNPs and functionalized GNPs to the DNA molecule led to increased configuration entropy. While conformational energy and van der Waals energy of the DNA molecule increased in the presence of the GNPs and functionalized GNPs, there was a reduction and an increase in the number of hydrogen bonds between complementary bases in the presence of the GNPs and functionalized GNPs, respectively. Radial distribution function was estimated for water molecules and sodium cations, compared to oxygen atoms of the phosphate group of the DNA molecule. Results were indicative of the release of water molecules from around the DNA molecule in the presence of the GNPs. In addition, the distance between sodium cations and the GNPs decreased. Nevertheless, no such phenomenon occurred in the presence of the functionalized GNPs. Therefore, according to results, it seems that GNPs decreased the stability of the DNA molecule and the functionalized GNPs with partial negative charge caused structural changes and created compression, but did not destroy the double-strand structure of the DNA molecule.     

NMR studies of DNA single strands and DNA:RNA hybrids with and without 1-propynylation at C5 of oligopyrimidines

The 1-propynylation at C5 of consecutive pyrimidines in DNA can enhance DNA:RNA hybrid stability at 37 degrees C by over 1 kcal/mol of substitution [Barnes, T. W., III; Turner, D. H. J. Am. Chem. Soc.2001, 123, 4107-4118]. To provide information on the structural consequences of propynylation, two-dimensional NMR spectroscopy was used to study the structures of several oligonucleotides. Intraresidue nuclear Overhauser effect spectroscopy cross peaks were observed at 30 degrees C and a 200 ms mixing time in the H6-H1' region for 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P)) (ssPrODN) but not for 5'(dCCUCCUU) (ssODN), suggesting preorganization of the propynylated single strand. NMR structures of the duplexes 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (PrODN:RNA), 5'(dCC(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (sPrODN1:RNA), and 5'(dCCUCCUU)3':3'(rGAGGAGGAAAU)5' (ODN:RNA) indicate that their global structures are almost identical. The NMR data, however, suggest that the 5'-end of sPrODN1:RNA is more dynamic than that of PrODN:RNA. In the propynylated duplexes, the propyne group stacks on the aromatic ring of the 5'-base and extends into the major groove. The results suggest that the increased stability of the propynylated duplexes is caused by preorganization of the propynylated single strand and different interactions in the double strand. The propynyl group provides volume exclusion, enhanced stacking, and possibly different solvation.     

Computational study of the stereochemistry of intramolecular carbolithiation of an alkene by a secondary alkyllithium: stereochemistry change caused by a single THF molecule of solvation

Theoretical calculations reveal that the 40:1 ratio of trans- to cis-2-methylcyclopentylmethyllithium formed in the cyclization of 6-lithio-1-heptene by intramolecular carbolithiation is due to steric crowding in the transition state for the cis-cyclization pathway when a single THF molecule complexes the lithium cation. In the absence of this specific solvation, the cis-cyclization pathway is predicted to be slightly favored.     

A new technique for membrane characterisation: direct measurement of the force of adhesion of a single particle using an atomic force microscope

An Atomic Force Microscope (AFM) has been used to quantify directly the adhesive force between a colloid probe and two polymeric ultrafiltration membranes of similar MWCO (4000 Da) but different materials (ES 404 and XP 117, PCI Membrane Systems (UK)). The colloid probe was made from a polystyrene sphere (diameter 11 μm) glued to a V shaped AFM cantilever. Measurements were made in 10⁻² M NaCl solution at pH 8. It was found that the adhesive force at the ES 404 membrane was more than five times greater than that at the XP 117 membrane. As it allows direct quantification of particle/membrane interactions, this technique should be invaluable in the development of new membrane materials and in the elucidation of process behaviour.