Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

<p>DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.</p>

Potential of Marine Terpenoids against SARS-CoV-2: An In Silico Drug Development Approach

In an emergency, drug repurposing is the best alternative option against newly emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, several bioactive natural products have shown potential against SARS-CoV-2 in recent studies. The present study selected sixty-eight broad-spectrum antiviral marine terpenoids and performed molecular docking against two novel SARS-CoV-2 enzymes (main protease or Mpro or 3CLpro) and RNA-dependent RNA polymerase (RdRp). In addition, the present study analysed the physiochemical-toxicity-pharmacokinetic profile, structural activity relationship, and phylogenetic tree with various computational tools to select the ‘lead’ candidate. The genomic diversity study with multiple sequence analyses and phylogenetic tree confirmed that the newly emerged SARS-CoV-2 strain was up to 96% structurally similar to existing CoV-strains. Furthermore, the anti-SARS-CoV-2 potency based on a protein−ligand docking score (kcal/mol) exposed that the marine terpenoid brevione F (−8.4) and stachyflin (−8.4) exhibited similar activity with the reference antiviral drugs lopinavir (−8.4) and darunavir (−7.5) against the target SARS−CoV−Mpro. Similarly, marine terpenoids such as xiamycin (−9.3), thyrsiferol (−9.2), liouvilloside B (−8.9), liouvilloside A (−8.8), and stachyflin (−8.7) exhibited comparatively higher docking scores than the referral drug remdesivir (−7.4), and favipiravir (−5.7) against the target SARS-CoV-2−RdRp. The above in silico investigations concluded that stachyflin is the most ‘lead’ candidate with the most potential against SARS-CoV-2. Previously, stachyflin also exhibited potential activity against HSV-1 and CoV-A59 within IC50, 0.16–0.82 µM. Therefore, some additional pharmacological studies are needed to develop ‘stachyflin’ as a drug against SARS-CoV-2.

Feruloyl Sucrose Esters: Potent and Selective Inhibitors of α-glucosidase and α-amylase.

Introduction: Feruloyl Sucrose Esters (FSEs) are a class of Phenylpropanoid Sucrose Esters (PSEs) widely distributed in plants. They were investigated as potential selective Alpha Glucosidase Inhibitors (AGIs) to eliminate the side effects associated with the current commercial AGIs. The latter effectively lowers blood glucose levels in diabetic patients but causes severe gastrointestinal side effects. Methods: Systematic structure-activity relationship (SAR) studies using in silico, in vitro and in vivo experiments were used to accomplish this aim. FSEs were evaluated for their in vitro inhibition of starch and oligosaccharide digesting enzymes α-glucosidase and α-amylase followed by in silico docking studies to identify the binding modes. A lead candidate, FSE 12 was investigated in an STZ mouse model. Results: All active FSEs showed desired higher % inhibition of α-glucosidase and desired lower inhibition of α-amylase in comparison to AGI gold standard acarbose. This suggests a greater selectivity of the FSEs towards α-glucosidase than α-amylase, which is proposed to eliminate the gastrointestinal side effects. From the in vitro studies, the position and number of the feruloyl substituents on the sucrose core, the aromatic ‘OH’ group, and the diisopropylidene bridges were key determinants of the % inhibition of α-glucosidase and α-amylase. In particular, the diisopropylidene bridges are critical for achieving inhibition selectivity. Molecular docking studies of the FSEs corroborates the in vitro results. The molecular docking studies further reveal that the presence of free aromatic ‘OH’ groups and the substitution at position 3 on the sucrose core are critical for the inhibition of both the enzymes. From the in vitro and molecular docking studies, FSE 12 was selected as a lead candidate for validation in vivo. The oral co-administration of FSE 12 with starch abrogated the increase in post-prandial glucose and significantly reduced blood glucose excursion in STZ-treated mice compared to control (starch only) mice. Conclusion: Our studies reveal the potential of FSEs as selective AGIs for the treatment of diabetes, with a hypothetical reduction of side effects associated with commercial AGIs.

2-chloro-5-(1-hydroxy-3-oxoisoindolin-1-yl)benzenesulfonamide Derivatives as Potential Urease Inhibitors: Synthesis, In-vitro and Molecular Modeling Approach

The present work is focused on the identification of 2-chloro-5-(1-hydroxy-3-oxoisoindolin-1-yl) benzenesulfonamides (5a-4k) Schiff bases as potent urease inhibitors. The Rationale behind the study was to introduce such lead candidate with lower micro-molar inhibitory values for the treatment of GIT disorders including gastric and peptic ulcer and hepatic encephalopathy. The synthesized compounds exhibited excellent anti-urease activity which was further supported by in-silico investigations. Therefore it can be suggested that these derivatives can be considered as a potential lead in future for synthesis of potent urease inhibitors.

Natural Bioactive Cyclopeptides from Microbes as Promising Anticancer Drug Leads: A Mini-review

Natural products from microbes are a rich source of bioactive molecules to serve as drug leads, predominantly in cancer therapy. Peptides are among the essential nature-derived biomolecules. Owing to their great diversity and favorable characteristics, cyclic peptides (cyclopeptides) from natural sources have become a propitious lead candidate for the development of therapeutics, including anticancer drugs. This present mini-review highlights cyclopeptides from microbial-derived natural products that have demonstrated significant cytotoxicity or anticancer activities. Moreover, this mini-review also provides a look into the mode of action of anticancer cyclopeptides. Selected examples are given for the potent anticancer cyclopeptides isolated in the recent decade from fungi and bacteria from both terrestrial and marine origins. Naturally occurring cyclopeptides with canonical and non-canonical amino acids isolated from fungi, myxobacteria, actinomycetes, marine cyanobacteria, and microbes associated with marine organisms and their anticancer activity are featured herein.

Super-Cationic Peptide Dendrimers—Synthesis and Evaluation as Antimicrobial Agents

Microbial infections are a major public health concern. Antimicrobial peptides (AMPs) have been demonstrated to be a plausible alternative to the current arsenal of drugs that has become inefficient due to multidrug resistance. Herein we describe a new AMP family, namely the super-cationic peptide dendrimers (SCPDs). Although all members of the series exert some antibacterial activity, we propose that special attention should be given to (KLK)2KLLKLL-NH2 (G1KLK-L2KL2), which shows selectivity for Gram-negative bacteria and virtually no cytotoxicity in HepG2 and HEK293. These results reinforce the validity of the SCPD family as a valuable class of AMP and support G1KLK-L2KL2 as a strong lead candidate for the future development of an antibacterial agent against Gram-negative bacteria.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.