Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis.

Aims: To develop a strategy to increase the stability of transcripts of structural genes expressed under the control of sacR, the leader region of Bacillus subtilis levansucrase gene. Methods and Results: Insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it was observed that the mRNA stability and the final protein production could be increased or decreased. Conclusions: This mRNA stabilization can be used to increase exocellular protein production in the degU32 (Hy) mutant. Significance and Impact of the Study: This approach can be applied to the expression of heterologous genes of biotechnological interest

Autogenous modulation of the Bacillus subtilis sacB-levB-yveA levansucrase operon by levB transcript.

Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression. Conversely, independent expression of levB greatly stimulates operon expression in Bacillus subtilis. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect. In vitro, the levB transcript displays an affinity to the N-terminal fragment of SacY (KD 0.2 µM), the regulatory protein that prevents transcription termination of levansucrase operon. This positive feed back loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology

L'opéron lévane saccharase de Bacillus subtilis (régulation transcriptionnelle et post-transcriptionnelle)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Discovery of an entropically-driven small molecule streptavidin binder from nucleic acid-encoded libraries

Dehydrocholic acid was identified as a selective streptavidin binder from a PNA-tagged library. Isothermal calorimetry titration measurements showed this interaction to be entropically driven. Peptides tagged with dehydrocholic acid can be captured on a streptavidin resin and released under thermal conditions

The RB596 antibody recognizes a linear epitope from the spike S protein from SARS-CoV-2

The recombinant antibody RB596 recognizes a linear epitope (residues 973-984, ISSVLNDILSRL) from the SARS-CoV-2 spike S protein

DNA-templated combinatorial assembly of small molecule fragments amenable to selection/amplification cycles

The discovery of small molecule probes which selectively modulate biological pathways is a cornerstone in the development of new therapeutics. Progress in our ability to access libraries of biologically relevant small molecules in conjunction with streamlined screening technologies have also enabled a more systematic approach to chemical biology. Nevertheless, the current state of the art still requires a large infrastructure and only a small fraction of the proteome has been addressed thus far. The emergence of technologies based on nucleic acid encoding of small molecules presents a new screening paradigm. We describe a method based on DNA-templated combinatorial display of PNA-encoded drug fragments affording 62500 combinations which can be amplified following a selection. This concept was demonstrated with a screen against a representative target, carbonic anhydrase, by iterative cycles of affinity selection, amplification of DNA template and “translation” back into selected library members. The results show a clear convergence towards combinations which, upon resynthesis as covalent adducts, proved to bind cooperatively to carbonic anhydrase

Experimental Identification of Immuno- dominant B-cell Epitopes from SARS-CoV-2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current public health crisis with devastating consequences to our societies. This COVID-19 pandemic has become the most serious threat to global public health in recent history. Given the unprecedented economic and social impact that it is causing, identification of immunodominant epitopes from SARS-CoV-2 is of great interest, not only to gain better insight into the adaptive immune response, but also for the development of vaccines, treatments and diagnostic tools. In this review, we summarize the already published or preprinted reports on the experimental identification of B-cell linear epitopes of SARS-CoV-2 proteins. Six different epitopes leading to neutralizing antibodies have been identified. Moreover, a summary of peptide candidates to be used for diagnostic tools is also included

DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

Fragment-based lead discovery has proven to be a powerful method in the drug discovery process. The combinatorial output that is accessible by combining fragments is very attractive; however, identifying fragment pairs that bind synergistically and linking them productively can be challenging. Several technologies have now been established to prepare and screen nucleic acid-encoded libraries (ssDNA, dsDNA, PNA), and it has been shown that pairs of molecules combined by hybridization can bind synergistically to a target. Herein we apply this concept to combinatorially pair two libraries of small molecule fragments, use the fittest fragments supplemented with closely related analogs to build a focused library covalently linking the fragments with different spacers, and apply this strategy to the discovery of a potent ligand for Hsp70

Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moieties

DNA-encoded chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2

Assembly of PNA-Tagged Small Molecules, Peptides, and Carbohydrates onto DNA Templates: Programming the Combinatorial Pairing and Inter-ligand Distance

The biochemical stability and desirable hybridization properties of peptide nucleic acids (PNA) coupled to the robustness of the peptidic chemistry involved in their oligomerization make them an attractive nucleic acid tag to encode molecules and program their assembly into higher order oligomers. The ability to program the dimerization of ligands with controlled distance between the ligands has important applications in emulating multimeric interactions. Additionally, the ability to program different permutations of ligand assemblies in a combinatorial fashion provides access to a broad diversity and offers a rapid screening method for fragment based approaches to drug discovery. Herein, we describe protocols to covalently link diverse carbohydrates, peptides, or small molecules to PNA and combinatorially assemble them in solution onto libraries of DNA templates or onto DNA microarrays using a commercial platform without recourse to specialized equipment or heavy upfront investment