Synthesis and Covalent Attachment of a Methylene Blue Derivative to A Triple Helix Forming Oligonucleotide - The Way to New Anticancer Drugs

Photoactive methylene blue (MB) is a cationic planar chromophore which is able to interact with double and triple helical DNA. Thus on its applications can b e used in the development of a new drug that specifically target a DNA sequence. Covalent attachment of the MB to the DNA through a flexible heptamethylene linker has been accomplished, enabling the defined positioning of the dye moiety at specific sites of the DNA strands by forming a triple helix. The structure of MB itself does not provide a suitable functional group for a direct attachment to an oligonucleotide. Thus, the MB derivative, 3-N-(4-carboxybutyl)-ethylamino-7-dimethylaminophenaza-thionumchloride was synthesized. The attachment of the MB derivative to a 3'-aminoalkylated oligonucleotide was accomplished by the activation of the carboxyl group with a yield from 40%-70%. A precipitation step using n-butanol was performed before HPLC purification in order to remove the excess of activated MB ester. Unreacted oligonucleotide was identified in the chromatogram from its absorbance at the typical wave length for nucleic acids and its lack of absorbance at 660 nm. The conjugate products bearing the MB moiety were recognized from the additional absorbance at around 660 nm. Another confirmation comes from the MS data. ID 'H NMR spectra of the MB-oligonucleotide conjugates as well as the non-conjugated 3'-aminoalkyl-oligonucleotide in H2O are acquired to compare the oligonucleotide before and after conjugation with MB

NMR studies on oligonucleotide – Methylene blue conjugates targeting double-helical nucleic acids

Methylene blue (MB) – nucleic acid interactions are of considerable interest due to the photosensitizing activity of the dye with potential applications in medicine and biotechnology. Covalent attachment of the MB to an oligonucleotide through a flexible heptamethylene linker enabled a positioning of the dye moiety to specific sites through triplex formation with a target duplex. NMR studies demonstrated interactions of MB with the nucleic acids. In sequences with the MB moiety facing the triplex-duplex junction with an alternating CG duplex overhang next to a T·A·T triple-helical tract, proton resonances experienced severe linebroadening upon MB binding and point to kinetically labile complexes with exchange among different binding modes. For sequences with the MB moiety facing a terminal T·A·T base triad of the triplex tract, structural heterogeneity decreased when compared to a triplex without MB attached to the third strand. Also, the thermal stability of the latter construct increased significantly in the presence of MB, indicating external end stacking as predominant binding mode. Without any obvious disruptions of sequential imino-imino NOE contacts within the triplex and duplex tracts, a most favorable intercalation between T·A·T base triples or CG base pairs is not supported by the present data under our experimental conditions

Thermal Denaturation Study on The Binding of A Methylene Blue Derivative to A Triple Helix Forming Oligonucleotide

Photoactive methylene blue(MB) is a cationic planar chromophore which is known to interact with double helical and triple helical DNA. UV spectroscopy have been employed to investigate the interaction between this dye and various DNA sequences, thus complementing the indirect data from LD, CD and/or fluorescence spectroscopic data available up to now. Covalent attachment of the MB to the DNA through a flexible heptamethylene linker has been accomplished in several steps, enabling the defined positioning of the dye moiety at specific sites of the DNA strands. UV thermal melting experiments indicate no significant stabilization/destabilization, demonstrated that MB interacts with all triplexforming DNA sequences studied here, albeit in different binding modes and strengths. In contrast, for sequences with the duplex overhang moved to the other side of the construct and with the MB moiety facing the terminal T-A.T triplex tract, imino resonances were noticeably sharper compared to the identical triplex sequence without MB. The corresponding triple helix-MB conjugate was stabilized through the dye-DNA interactions by up to IO°C as indicated by the UV melting data....file abstrak http://repository.ubaya.ac.id/7096/2/Thermal%20Denaturation%20Study_Abstract_2013.pd

Structural Aspects of Split G-Quadruplexes in Quadruplex-Duplex Hybrid Systems

G-quadruplexes are secondary structures of nucleic acids increasingly employed for biological and also technological applications. Being of particular interest, a structural motif called quadruplex-duplex (QD) hybrid comprises both a G-quadruplex and duplex domain. Here, we attempted to engineer a bimolecular quadruplex-duplex hybrid through dual recognition of a receptor by a target strand with the simultaneous formation of both G-quadruplex and duplex structures. Three different constructs were designed for the G-quadruplex receptor, featuring either one, two, or three vacant sites. Addition of the target strand filled the vacancies when simultaneously hybridizing with flanking sequences to form a duplex as shown by NMR studies. Formed QD hybrid constructs either constitute an 11 : 1, 10 : 2, or 9 : 3 split G-quadruplex system. The 10 : 2 QD construct folds into a non-canonical V-loop topology. Remarkably, the latter also accommodates 5’-overhang residues of the target strand although suggested to impose a steric penalty. Formation of the duplex domain is demonstrated to be critical for the successful formation of the intact G-quadruplex domain. With the formation of a unique QD junction for detection, the constructs may constitute valuable tools for single strand capturing strategies

Identification of DNA G–quadruplex Forming Sequence in Shrimp White Spot Syndrome Virus (WSSV)

White spot syndrome virus (WSSV) is considered one of the most infectious and lethal viruses that affect shrimp. Bioinformatic studies revealed several G–quadruplex forming sequences at the open reading frame region. Moreover, the sequences are widely conserved through all deposited WSSV sequences. Introductory structural studies on two sequences, namely WSSV131 and WSSV172, are proposed to form a quadruplex. While WSSV172 forms a mixture of quadruplex topologies, WSSV131 is suggested to form a parallel topology, as indicated by the NMR spectra and circular dichroism (CD) ellipticity pattern. CD spectra also suggested that the major parallel species of the WSSV131 sequence are found to be stable above 60 °C. Ultimately, these results may open a new strategy for WSSV treatment by targeting the quadruplex confirmation with a quadruplex binding ligand

Carbon footprints of cities and other human settlements in the UK

A growing body of literature discusses the CO2 emissions of cities. Still, little is known about emission patterns across density gradients from remote rural places to highly urbanized areas, the drivers behind those emission patterns and the global emissions triggered by consumption in human settlements—referred to here as the carbon footprint. In this letter we use a hybrid method for estimating the carbon footprints of cities and other human settlements in the UK explicitly linking global supply chains to local consumption activities and associated lifestyles. This analysis comprises all areas in the UK, whether rural or urban. We compare our consumption-based results with extended territorial CO2 emission estimates and analyse the driving forces that determine the carbon footprint of human settlements in the UK. Our results show that 90% of the human settlements in the UK are net importers of CO2 emissions. Consumption-based CO2 emissions are much more homogeneous than extended territorial emissions. Both the highest and lowest carbon footprints can be found in urban areas, but the carbon footprint is consistently higher relative to extended territorial CO2 emissions in urban as opposed to rural settlement types. The impact of high or low density living remains limited; instead, carbon footprints can be comparatively high or low across density gradients depending on the location-specific socio-demographic, infrastructural and geographic characteristics of the area under consideration. We show that the carbon footprint of cities and other human settlements in the UK is mainly determined by socio-economic rather than geographic and infrastructural drivers at the spatial aggregation of our analysis. It increases with growing income, education and car ownership as well as decreasing household size. Income is not more important than most other socio-economic determinants of the carbon footprint. Possibly, the relationship between lifestyles and infrastructure only impacts carbon footprints significantly at higher spatial granularity

Physical and virtual carbon metabolism of global cities

Urban activities have profound and lasting effects on the global carbon balance. Here we develop a consistent metabolic approach that combines two complementary carbon accounts, the physical carbon balance and the fossil fuel-derived gaseous carbon footprint, to track carbon coming into, being added to urban stocks, and eventually leaving the city. We find that over 88% of the physical carbon in 16 global cities is imported from outside their urban boundaries, and this outsourcing of carbon is notably amplified by virtual emissions from upstream activities that contribute 33–68% to their total carbon inflows. While 13–33% of the carbon appropriated by cities is immediately combusted and released as CO₂, between 8 and 24% is stored in durable household goods or becomes part of other urban stocks. Inventorying carbon consumed and stored for urban metabolism should be given more credit for the role it can play in stabilizing future global climate

Physical and virtual carbon metabolism of global cities

Urban activities have profound and lasting effects on the global carbon balance. Here we develop a consistent metabolic approach that combines two complementary carbon accounts, the physical carbon balance and the fossil fuel-derived gaseous carbon footprint, to track carbon coming into, being added to urban stocks, and eventually leaving the city. We find that over 88% of the physical carbon in 16 global cities is imported from outside their urban boundaries, and this outsourcing of carbon is notably amplified by virtual emissions from upstream activities that contribute 33–68% to their total carbon inflows. While 13–33% of the carbon appropriated by cities is immediately combusted and released as CO2, between 8 and 24% is stored in durable household goods or becomes part of other urban stocks. Inventorying carbon consumed and stored for urban metabolism should be given more credit for the role it can play in stabilizing future global climate

Complete O(v^2) corrections to the static interquark potential from SU(3) gauge theory

For the first time, we determine the complete spin- and momentum-dependent order v^2 corrections to the static interquark potential from simulations of QCD in the valence quark approximation at inverse lattice spacings of 2-3 GeV. A new flavor dependent correction to the central potential is found. We report a 1/r^2 contribution to the long range spin-orbit potential V_1'. The other spin-dependent potentials turn out to be short ranged and can be well understood by means of perturbation theory. The momentum-dependent potentials qualitatively agree with minimal area law expectations. In view of spectrum calculations, we discuss the matching of the effective nonrelativistic theory to QCD as well as renormalization of lattice results. In a first survey of the resulting bottomonia and charmonia spectra we reproduce the experimental levels within average errors of 12.5 MeV and 22 MeV, respectively.Comment: 54 pages REVTeX with 24 encapsuled ps figure