Design, synthesis, conformational analysis and nucleic acid hybridisation properties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM)

Pyrrolidine-amide oligonucleotide mimics (POM) 1 were designed to be stereochemically and conformationally similar to natural nucleic acids, but with an oppositely charged, cationic backbone. Molecular modelling reveals that the lowest energy conformation of a thymidyl-POM monomer is similar to the conformation adopted by ribonucleosides. An e cient solution phase synthesis of the thymidyl POM oligomers has been developed, using both N-alkylation and acylation coupling strategies. 1H NMR spectroscopy con rmed that the highly water soluble thymidyl-dimer, T2-POM, preferentially adopts both a con guration about the pyrrolidine N-atom and an overall conformation in D2O that are very similar to a typical C3 -endo nucleotide in RNA. In addition the nucleic acid hybridisation properties of a thymidyl-pentamer, T5-POM, with an N-terminal phthalimide group were evaluated using both UV spectroscopy and surface plasmon resonance (SPR). It was found that T5-POM exhibits very high a nity for complementary ssDNA and RNA, similar to that of a T5-PNA oligomer. SPR experiments also showed that T5-POM binds with high sequence delity to ssDNA under near physiological conditions. In addition, it was found possible to attenuate the binding a nity of T5-POM to ssDNA and RNA by varying both the ionic strength and pH. However, the most striking feature exhibited by T5-POM is an unprecedented kinetic binding selectivity for ssRNA over DNA

Merging enzymes with chemocatalysis for amide bond synthesis

Amides are one of the most fundamental chemical bonds in nature. In addition to proteins and other metabolites, many valuable synthetic products comprise amide bonds. Despite this, there is a need for more sustainable amide synthesis. Herein, we report an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds. This synergistic one-pot combination of chemo- and biocatalysis provides an amide bond disconnection to precursors, that are orthogonal to those in classical amide synthesis, obviating the need for protecting groups and delivering amides in a manner unachievable using existing catalytic regimes. Our integrated approach also affords broad scope, very high (molar) substrate loading, and has excellent functional group tolerance, telescoping routes to natural product derivatives, drug molecules, and challenging chiral amides under environmentally friendly conditions at scale

Dual transcriptional-translational cascade permits cellular level tuneable expression control.

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems

Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli

Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians’ diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli–compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs—e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer—as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion

The snomipede : a parallel platform for scanning near-field photolithography.

Using scanning near-field lithography (SNP), it is possible to pattern molecules at surfaces with a resolution as good as 9 nm [M. Montague, R. E. Ducker, K. S. L. Chong, R. J. Manning, F. J. M. Rutten, M. C. Davies and G. J. Leggett, Langmuir 23 (13), 7328–7337 (2007)]. However, in common with other scanning probe techniques, SNP has previously been considered a serial process, hindering its use in many applications. IBM’s “Millipede” addresses this problem by utilizing an array of local probes operating in parallel. Here, we describe the construction of two instruments (Snomipedes) that integrate near-field optical methods into the parallel probe paradigm and promise the integration of top–down and bottom–up fabrication methods over macroscopic areas. Both are capable of performing near-field lithography with 16 probes in parallel spanning approximately 2 mm. The instruments can work in both ambient and liquid environments, key to many applications in nanobiology. In both, separate control of writing is possible for each probe. We demonstrate the deprotection of self-assembled monolayers of alkylsilanes with photocleavable protecting groups and subsequent growth of nanostructured polymer brushes from these nanopatterned surfaces by atom-transfer radical polymerization

Gene editing enables rapid engineering of complex antibiotic assembly lines

From Springer Nature via Jisc Publications RouterHistory: received 2021-09-09, accepted 2021-11-02, registration 2021-11-08, pub-electronic 2021-11-25, online 2021-11-25, collection 2021-12Publication status: PublishedFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/L013754/1, BB/N023536/1Abstract: Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized

Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4.0–6.6 kcal/mol. Slight destabilization, by ∼0.5–1.5 kcal/mol, is observed for 5′- or 3′-terminal UNA residues. Furthermore, thermodynamic effects caused by UNA residues are extremely additive with ΔG°37 conformity up to 98%. Direct mismatches involving UNA residues decrease the thermodynamic stability less than unmodified mismatches in RNA duplexes. Additionally, the presence of UNA residues adjacent to unpaired RNA residues reduces mismatch discrimination. Thermodynamic analysis of UNA 5′- and 3′-dangling ends revealed that stacking interactions of UNA residues are always less favourable than that of RNA residues. Finally, circular dichroism spectra imply no changes in overall A-form structure of UNA–RNA/RNA duplexes relative to the unmodified RNA duplexes

A versatile method for the preparation of conjugates of peptides with DNA/PNA/analog by employing chemo-selective click reaction in water

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ∼3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products