{ "items": [ "\n\n
We report the controlled release of immobilized cholesteryl-tetraethyleneglycol-DNA (chol-DNA) from micropatterned SU-8 surfaces by a spreading lipid film. The release of chol-DNA is rapid and on the order of the spreading rate of the lipid film beta = 1-3 microm2/s ( approximately 10(5) molecules of DNA per second). The lipid film serves as a poor solvent for the DNA adduct, which upon contact redistributes into the aqueous phase. Thus, the release of DNA is accompanied by a change in surface hydrophobicity. The method can be used for creating arbitrary concentration profiles of DNA in solution over time or to dynamically change surface properties on demand in, for example, micro- and nanofluidic devices. Examples of DNA release from spiral, comb, meander, and triangular as well as from nanoscale SU-8 lanes are shown.
\n \n\n \n \nThis article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC(O), is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC(O) is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (epsilon = 9000 M(-1) cm(-1)) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tC(O) shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC(O), on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC(O) a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).
\n \n\n \n \nThe CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covalently linking presynthesized RNA strands with high efficiency (trans-ligation). In an alternative strategy, intrastrand click ligation was employed to produce a functional hammerhead ribozyme containing a novel nucleic acid backbone mimic at the catalytic site (cis-ligation). The ability to synthesize long RNA strands by a combination of solid-phase synthesis and click ligation is an important addition to RNA chemistry. It is compatible with a plethora of site-specific modifications and is applicable to the synthesis of many biologically important RNA molecules.
\n \n\n \n \nA new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.
\n \n\n \n \nThe crystal structure of the oligonucleotide d(CGCAAATTO8GGCG), containing the chemically modified base 8-hydroxydeoxyguanine (O8G), has been determined at 2.5-A resolution and refined to a crystallographic R-factor of 16.8%. The B-type DNA helix contains standard Watson-Crick base pairs except at the mismatch sites, where O8G adopts a syn conformation and forms hydrogen bonds to adenine in the anti conformation. The thermodynamic stability of the duplex was found by UV melting techniques to be intermediate between the native oligonucleotide d(CGCAAATTTGCG) and an oligonucleotide containing A.G mispairs d(CGCAAATTGGCG). Comparison of the structure of the O8G(syn).A(anti) base pair with those of Watson-Crick base pairs has given a reason why O8G.A base pairs are not well repaired by DNA proofreading enzymes.
\n \n\n \n \nDinucleoside phosphoramidites containing a triazole internucleotide linkage flanked by locked nucleic acid (LNA) were synthesized and incorporated into oligonucleotides (ONs). ONs bearing both LNA and triazole at multiple sites were obtained and their biophysical properties including enzymatic stability and binding affinity for RNA and DNA targets were studied. t-LNAs with four incorporations of a dinucleoside monomer having LNA on either side of the triazole linkage bind to their RNA target with significantly higher affinity and greater specificity than unmodified oligonucleotides, and are remarkably stable to nuclease degradation. A similar but reduced effect on enzymatic stability and binding affinity was noted for LNA only on the 3'-side of the triazole linkage. Thus, by combining unnatural triazole linkages and LNA in one unit (t-LNA), we produced a promising class of ONs with reduced anionic charge and potential for antisense applications.
\n \n\n \n \nFormaldehyde is produced in cells by enzyme-catalysed demethylation reactions, including those occurring on N-methylated nucleic acids. Formaldehyde reacts with nucleobases to form N-hydroxymethylated adducts that may contribute to its toxicity/carcinogenicity when added exogenously, but the chemistry of these reactions has been incompletely defined. We report NMR studies on the reactions of formaldehyde with canonical/modified nucleobases. The results reveal that hydroxymethyl hemiaminals on endocyclic nitrogens, as observed with thymidine and uridine monophosphates, are faster to form than equivalent hemiaminals on exocyclic nitrogens; however, the exocyclic adducts, as formed with adenine, guanine and cytosine, are more stable in solution. Nucleic acid demethylase (FTO)-catalysed hydroxylation of (6-methyl)adenosine results in (6-hydroxymethyl)adenosine as the major observed product; by contrast no evidence for a stable 3-hydroxymethyl adduct was accrued with FTO-catalysed oxidation of (3-methyl)thymidine. Collectively, our results imply N-hydroxymethyled adducts of nucleic acid bases, formed either by reactions with formaldehyde or via demethylase catalysis, have substantially different stabilities, with some being sufficiently stable to have functional roles in disease or the regulation of nucleic acid/nucleobase activity.
\n \n\n \n \nThe copper-catalyzed azide-alkyne cycloaddition reaction has been used for the template-mediated chemical ligation of two oligonucleotide strands, one with a 5'-alkyne and the other with a 3'-azide, to produce a DNA strand with an unnatural backbone at the ligation point. A template-free click-ligation reaction has been used for the intramolecular circularization of a single stranded oligonucleotide which was used as a template for the synthesis of a covalently closed DNA catenane.
\n \n\n \n \nThe design of nanoparticles that can selectively perform multiple roles is of utmost importance for the development of the next generation of nanoparticulate drug delivery systems. So far most research studies are focused on the customization of nanoparticulate carriers to maximize their drug loading, enhance their optical signature for tracking in cells or provide photo-responsive effects for therapeutic purposes. However, a vital requirement of the new generation of drug carriers must be the ability to deliver their payload selectively only to cells of interest rather than the majority of various cells in the vicinity. Here we show for the first time a new design of nanoparticulate drug carriers that can specifically distinguish different cell types based on their mRNA signature. These nanoparticles sense and efficiently kill model tumour cells by the delivery of an anti-cancer drug but retain their payload in cells lacking the specific mRNA target.
\n \n\n \n \nA label-free, surface-enhanced Raman spectroscopy-based assay for detecting DNA hybridization at an electrode surface and for distinguishing between mutations in DNA is demonstrated. Surface-immobilized DNA is exposed to a binding agent that is selective for dsDNA and acts as a reporter molecule. Upon application of a negative potential, the dsDNA denatures into its constituent strands, and the changes in the spectra of the reporter molecule are monitored. This method has been used to distinguish between a wild-type, 1653C/T single-point mutation and \u0394F508 triplet deletion in the CFTR gene. The use of dsDNA-selective binding agents as reporter molecules in a discrimination assay removes the burden of synthetically modifying the target to be detected, while retaining flexibility in the choice of the reporter molecule.
\n \n\n \n \nWe demonstrate a new method to reversibly cross-link DNA-nanoparticle dimers, trimers, and tetramers using light as an external stimulus. A DNA interstrand photo-cross-linking reaction is possible via ligation of a cyano-vinyl carbazole nucleoside with an opposite thymine when irradiated at 365 nm. This reaction results in nanoparticle assemblies that are not susceptible to DNA dehybridization conditions. The chemical bond between the two complementary DNA strands can be reversibly broken upon light irradiation at 312 nm. This is the first example of reversible ligation in DNA-nanoparticle assemblies using light and enables new developments in the field of programmed nanoparticle organization.
\n \n\n \n \nDNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.
\n \n\n \n \nA DNA strand containing a triazole phosphodiester mimic is an efficient template for in vitro transcription. This is the first demonstration of transcription through a heavily modified DNA backbone linkage and it suggests that click-ligated DNA could be useful for the direct synthesis of biologically active RNA and proteins.
\n \n\n \n \nSynthetic chemistry has been central to the design of modern methods of genetic analysis. In this article, we discuss the underlying chemistry and biophysical principles that have been used in the development of robust methods for the analysis of DNA in the diagnostic laboratory.
\n \n\n \n \nWe have prepared oligonucleotides with a naphthylquinoline triplex-binding ligand covalently tethered to the 5'-end and have used UV-melting and DNase I footprinting to examine the stability of intra- and inter-molecular triplexes containing this modification. We find that covalent attachment of the ligand increases the melting temperature of intramolecular 6-mer triplexes by about 14 K, and increases the binding of 9-mer oligonucleotides to their duplex target sites by about 60-fold.
\n \n\n \n \nWe have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption.
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