1,720,973 research outputs found
Design and synthesis of a biotinylated chemical probe for detecting the molecular targets of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin
No full textPseudomonas aeruginosa is a human pathogen associated with a variety of life-threatening nosocomial infections. This organism produces a range of virulence factors which actively cause damage to host tissues. One such virulence factor is pyocyanin, known to play a crucial role in the pathogenesis of P. aeruginosa infections. Previous studies had identified a novel compound capable of strongly inhibiting the production of pyocyanin. It was postulated that this inhibition results from modulation of an intercellular communication system termed quorum sensing, via direct binding of the compound with the LasR protein receptor. This raised the possibility that the compound could be an antagonist of quorum sensing in P. aeruginosa, which could have important implications as this intercellular signaling mechanism is known to regulate many additional facets of P. aeruginosa pathogenicity. However, there was no direct evidence for the binding of the active compound to LasR (or any other targets). Herein we describe the design and synthesis of a biotin-tagged version of the active compound. This could potentially be used as an affinity-based chemical probe to ascertain, in a direct fashion, the active compound’s macromolecular biological targets, and thus better delineate the mechanism by which it reduces the level of pyocyanin production
Expanding the chemical functionality of DNA nanomaterials generated by rolling circle amplification
Full text linkRolling circle amplification (RCA) is a powerful tool for the construction of DNA nanomaterials such as hydrogels, high-performance scaffolds and DNA nanoflowers (DNFs), hybrid materials formed of DNA and magnesium pyrophosphate. Such DNA nanomaterials have great potential in therapeutics, imaging, protein immobilisation, and drug delivery, yet limited chemistry is available to expand their functionality. Here, we present orthogonal strategies to produce densely modified RCA products and DNFs. We provide methods to selectively modify the DNA component and/or the protein cargo of these materials, thereby greatly expanding the range of chemical functionalities available to these systems. We have used our methodology to construct DNFs bearing multiple surface aptamers and peptides capable of binding to cancer cells that overexpress the HER2 oncobiomarker, demonstrating their potential for diagnostic and therapeutic applications
Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation
No full textCyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using ϕ-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by ϕ-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates
Searching for the ideal triazole: Investigating the 1,5-triazole as a charge neutral DNA backbone mimic
No full textA novel triazole linkage that mimics the phosphodiester backbone in DNA was designed, synthesised and evaluated. Unlike previous work which utilised copper to form a 1,4 triazole linkage in the DNA backbone, a ruthenium catalyst was used to yield a 1,5 triazole. The artificial linkage was incorporated into a DNA backbone via a phosphoramidite building block using solid phase synthesis. The biophysical properties of DNA with a 1,5 triazole linkage in the backbone were evaluated by UV melting and circular dichroism and compared to DNA modified with previously reported 1,4 triazole linkages of various lengths
Covalently attached intercalators restore duplex stability and splice-switching activity to triazole-modified oligonucleotides
Full text linkOligonucleotides are rapidly emerging as powerful therapeutics for hard to treat diseases. Short single-stranded oligonucleotides can base pair with target RNA and alter gene expression, providing an attractive therapeutic approach at the genetic level. Whilst conceptually appealing, oligonucleotides require chemical modification for clinical use. One emerging approach is to substitute the phosphodiester backbone with other chemical linkages such as triazole. The triazole linkage is inherently resistant to enzymatic degradation, providing stability in vivo, and is uncharged, potentially improving cell-penetration and in vivo distribution. Triazole linkages, however, are known to reduce RNA target binding affinity. Here we show that by attaching pyrene or anthraquinone to the ribose sugar on the 5′-side of the triazole, it is possible to recover duplex stability and restore the splice switching ability of triazole-containing oligonucleotides
A new Pseudomonas quinolone signal (PQS) binding partner: MexG
No full textThe opportunistic pathogen Pseudomonas aeruginosa utilises the cell–cell signalling mechanism known as quorum sensing to regulate virulence. P. aeruginosa produces two quinolone-based quorum sensing signalling molecules; the Pseudomonas quinolone signal (PQS) and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ). To date, only one receptor (the PqsR protein) has been identified that is capable of binding PQS and HHQ. Here, we report on the synthesis of PQS and HHQ affinity probes for chemical proteomic studies. The PQS affinity probe very effectively captured PqsR in vitro. In addition, we also identified an interaction between PQS and the “orphan” RND efflux pump protein, MexG. The PQS–MexG interaction was further confirmed by purifying MexG and characterizing its ability to bind PQS and HHQ in vitro. Our findings suggest that PQS may have multiple binding partners in the cell and provide important new tools for studying quinolone signalling in P. aeruginosa and other organisms
Consecutive 5′- and 3′-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response
No full textAntisense oligonucleotides are now entering the clinic for hard-to-treat diseases. New chemical modifications are urgently required to enhance their drug-like properties. We combine amide coupling with standard oligonucleotide synthesis to assemble backbone chimera gapmers that trigger an efficient RNase H response while improving serum life time and cellular uptake
Preparation and characterisation of manganese, cobalt and zinc DNA nanoflowers with tuneable morphology, DNA content and size
Full text linkRecently reported DNA nanoflowers are an interesting class of organic-inorganic hybrid materials which are prepared using DNA polymerases. DNA nanoflowers combine the high surface area and scaffolding of inorganic Mg2P2O7 nanocrystals with the targeting properties of DNA, whilst adding enzymatic stability and enhanced cellular uptake. We have investigated conditions for chemically modifying the inorganic core of these nanoflowers through substitution of Mg2+ with Mn2+, Co2+ or Zn2+ and have characterised the resulting particles. These have a range of novel nanoarchitectures, retain the enzymatic stability of their magnesium counterparts and the Co2+ and Mn2+ DNA nanoflowers have added magnetic properties. We investigate conditions to control different morphologies, DNA content, hybridisation properties, and size. Additionally, we show that DNA nanoflower production is not limited to Ф29 DNA polymerase and that the choice of polymerase can influence the DNA length within the constructs. We anticipate that the added control of structure, size and chemistry will enhance future application
Chemically modified nucleic acids and DNA intercalators as tools for nanoparticle assembly
No full textThe self-assembly of inorganic nanoparticles to larger structures is of great research interest as it allows the fabrication of novel materials with collective properties correlated to the nanoparticles' individual characteristics. Recently developed methods for controlling nanoparticle organisation have enabled the fabrication of a range of new materials. Amongst these, the assembly of nanoparticles using DNA has attracted significant attention due to the highly selective recognition between complementary DNA strands, DNA nanostructure versatility, and ease of DNA chemical modification. In this review we discuss the application of various chemical DNA modifications and molecular intercalators as tools for the manipulation of DNA-nanoparticle structures. In detail, we discuss how DNA modifications and small molecule intercalators have been employed in the chemical and photochemical DNA ligation in nanostructures; DNA rotaxanes and catenanes associated with reconfigurable nanoparticle assemblies; and DNA backbone modifications including locked nucleic acids, peptide nucleic acids and borane nucleic acids, which affect the stability of nanostructures in complex environments. We conclude by highlighting the importance of maximising the synergy between the communities of DNA chemistry and nanoparticle self-assembly with the aim to enrich the library of tools available for the manipulation of nanostructures.</p
The Pseudomonas Quinolone Signal (PQS)
No full textPseudomonas aeruginosa is an opportunistichuman pathogen that routinely appears near the top ofpublic health threat lists worldwide. P. aeruginosa causes in-fections by secreting a wealth of exceptionally active exo-products, leading to tissue damage. The synthesis of manyof these virulence factors is now known to be under the con-trol of the quorum sensing (QS) system. Over the last15 years, the Pseudomonas quinolone signal (PQS) has beenfound to play a crucial role in QS by linking the two seg-ments (las and rhl) of the P. aeruginosa N-acylhomoserinelactone-dependent QS signaling pathways. Herein, we pres-ent the discovery and elucidation of PQS signaling froma historical perspective, and also outline some of the out-standing research questions that still need to be addressed.Finally, we show how a better understanding of the bio-chemistry underpinning this pathway is leading to the devel-opment of new antimicrobial interventions with clear thera-peutic potential
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