Biology subjects

Pinheiro, V. B.

Publications and source records attributed to Pinheiro, V. B..

9 recordsLinked to original sources

bioRxiv2026

Modular click-chemistry-based engineering minimally perturbs CRISPR ribonucleoprotein formation for genome imaging applications

CRISPR-Cas9 has become a key genome imaging tool, because of its sequence-specific binding to genomic DNA. To achieve signal amplification or multiplexing, various complex CRISPR-Cas9 modifications were introduced, primarily relying on expression-based delivery. However, these engineering strategies often invoke structural perturbations and their performance remained dictated by vector design and in vivo CRISPR ribonucleoprotein (RNP) assembly. To date, research mainly focused on optimizing the expression-based delivery mechanisms of CRISPR components rather than addressing the engineering itself. Here, we developed a modular SPAAC (bio-orthogonal strain-promoted azide-alkyne cycloaddition) click-chemistry-based strategy for engineering the CRISPR RNP that is completely dissociated from its assembly. We demonstrated that CRISPR can be modified (with fluorophores and DNA) at all stages of RNP in vitro assembly and subsequently validated the technology for genome imaging. Importantly, we also revealed that engineering native CRISPR RNP only after it is being formed, minimizes structural perturbations to the complex and yields a two-fold enhanced accessibility of 'clicked' DNA. Finally, we utilized SPAAC to conjugate non-native RNA-DNA hybrids, regardless of 5'-3' directionality, to develop a covalently-attached rolling-circle signal amplification approach. The modularity and universal nature of our approach holds the potential to deliver significant advances to both CRISPR imaging and prime-editing fields.

bioengineering↗

bioRxiv2024

Navigating directed evolution efficiently: optimizing selection conditions and selection output analysis

Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acids (XNAs) synthetases and reverse transcriptases (RTs) capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

synthetic biology↗

bioRxiv2023

Escherichia coli Microcin B17 as a chassis for the development of novel antimicrobial peptides.

Withdrawal statementThe authors have withdrawn this manuscript because bioactivity of the reported peptides cannot be confirmed. Scaling up peptide synthesis bioproduction failed to reproduce the original bioactivity profile of the reported microcins (despite appropriate controls). At present, we believe that, in the original bioproduction platform, antibiotics (used in the upstream bacterial growth) co-purified with the microcin - accounting for or enhancing the bioactivity of the isolated peptides. Therefore, the authors do not wish this work to be cited as reference for the project. The DNA assembly platform remains valid and will be reported elsewhere. If you have any questions, please contact the corresponding author.

synthetic biology↗

bioRxiv2022

Directed evolution of mesophilic HNA polymerases providing insight into DNA polymerase mechanisms

Detailed biochemical characterization of natural and mutant enzymes provides essential clues to understand their mechanisms. There are, however, limits to the throughput of such approaches and they are not without errors. DNA polymerases have benefited from over 50 years of detailed study and remain not fully understood. As such, methods that allow high-throughput interrogation of variants, and viable analysis pipelines to identify relevant variants, become an important tool to accelerate research. Using the DNA polymerase from B. subtilis Phi29 bacteriophage as a model, we demonstrate how coupling focused libraries, selection and deep sequencing can be combined to identify variants of interest for characterization. As selection parameters can be controlled, different areas of an enzymes mechanism can be explored. Focusing selection on faster HNA (1,5-anhydrohexitol nucleic acid) synthesis, we identified P562del as a variant of interest, enriching significantly between rounds. Characterization confirmed its faster HNA synthesis initiation but lower processivity and fidelity. P562 is a non-conserved residue, unlikely to be selected by more traditional approaches, but its deletion recapitulates knowledge on how Phi29 exonuclease, thumb and TPR2 subdomains regulate polymerase function. Our data further support the hypothesis that Phi29 shows a two-state binding to its template: a fast non-replicative complex that transitions to a replication-competent state.

biochemistry↗

bioRxiv2022

Lira: Rotational Invariant Shape and ElectrostaticDescriptors for Small Molecules and ProteinPockets based on Real Spherical Harmonics

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSMotivationC_ST_ABSModern AI-based tools are increasing the number of protein structures available, creating an opportunity and a challenge for automated high-throughput drug discovery pipelines. The amount of data is overwhelming for the current methods, thus demanding new high-performance approaches for Machine Learning-based rational drug design. As shape and electrostatics are the main components for understanding protein-ligand interaction; they are the primary targets for efficient AI-compatible descriptors and their associated comparison methods. ResultsThe Lira toolbox is a set of components devised for describing, comparing and analysing shape and electrostatics for small ligands, peptides and protein pockets. It can generate databases with descriptors for tens of millions of shapes in a few hours, which can then be queried in seconds. The Lira design, focused on performance and reliability, makes its integration into AI-driven rational drug design pipelines simple. Availability and implementationLira packages, available for download at https://pinheirolab.com/, are free to use for research and educational purposes.

bioinformatics↗

bioRxiv2021

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population - a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements.

synthetic biology↗

bioRxiv2020

Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes

Although directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology, it is limited by the robustness and flexibility of available selection platforms - traditionally designed around a single desired function with limited scope for alternative applications. We report SNAP as a quantitative reporter for bacterial cell display, which enabled fast troubleshooting and systematic development of the selection platform. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed towards specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes. Together, this establishes bacterial display as a viable route towards the systematic engineering of all ligands and enzymes required for the development of XNA molecular biology.

synthetic biology↗

bioRxiv2019

Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

Protein engineering through directed evolution facilitates the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries with minimal hands-on time and error-free, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

bioengineering↗

bioRxiv2017

Efficiently Exploring Functional Space In Loop Engineering With Variations In Length And Composition

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with no available platforms capable of systematically generating or analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 {beta}-lactamase {Omega}-loop, involved in substrate specificity, identifying multiple novel extended spectrum {beta}-lactamases with loops of modified length and composition areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

bioengineering↗