RÉSUMÉ
Data science is playing an increasingly important role in the design and analysis of engineered biology. This has been fueled by the development of high-throughput methods like massively parallel reporter assays, data-rich microscopy techniques, computational protein structure prediction and design, and the development of whole-cell models able to generate huge volumes of data. Although the ability to apply data-centric analyses in these contexts is appealing and increasingly simple to do, it comes with potential risks. For example, how might biases in the underlying data affect the validity of a result and what might the environmental impact of large-scale data analyses be? Here, we present a community-developed framework for assessing data hazards to help address these concerns and demonstrate its application to two synthetic biology case studies. We show the diversity of considerations that arise in common types of bioengineering projects and provide some guidelines and mitigating steps. Understanding potential issues and dangers when working with data and proactively addressing them will be essential for ensuring the appropriate use of emerging data-intensive AI methods and help increase the trustworthiness of their applications in synthetic biology.
RÉSUMÉ
Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.
Sujet(s)
Conception assistée par ordinateur , Apprentissage profond , Peptides , Protéines , Techniques de biocapteur , Diffusion , Glucagon/composition chimique , Glucagon/métabolisme , Mesures de luminescence , Spectrométrie de masse , Hormone parathyroïdienne/composition chimique , Hormone parathyroïdienne/métabolisme , Peptides/composition chimique , Peptides/métabolisme , Structure secondaire des protéines , Protéines/composition chimique , Protéines/métabolisme , Spécificité du substrat , Modèles moléculairesRÉSUMÉ
Synthetic Biology has overcome many of the early challenges facing the field and is entering a systems era characterized by adoption of Design-Build-Test-Learn (DBTL) approaches. The need for automation and standardization to enable reproducible, scalable, and translatable research has become increasingly accepted in recent years, and many of the hardware and software tools needed to address these challenges are now in place or under development. However, the lack of connectivity between DBTL modules and barriers to access and adoption remain significant challenges to realizing the full potential of lab automation. In this review, we characterize and classify the state of automation in synthetic biology with a focus on the physical automation of experimental workflows. Though fully autonomous scientific discovery is likely a long way off, impressive progress has been made toward automating critical elements of experimentation by combining intelligent hardware and software tools. It is worth questioning whether total automation that removes humans entirely from the loop should be the ultimate goal, and considerations for appropriate automation versus total automation are discussed in this light while emphasizing areas where further development is needed in both contexts.
Sujet(s)
Laboratoire automatique , Biologie synthétique , Humains , Automatisation , Logiciel , Normes de référence , Plan de rechercheRÉSUMÉ
The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we achieve single-amino acid level sensitivity, enabling sequencing of combinations of amino acid substitutions across long protein strands. For greater sequencing accuracy, we demonstrate the ability to reread individual protein molecules, spanning hundreds of amino acids in length, multiple times, and explore the potential for high accuracy protein barcode sequencing. Further, we develop a biophysical model that can simulate raw nanopore signals a priori, based on amino acid volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine intact, folded protein domains for complete end-to-end analysis. These results provide proof-of-concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.
RÉSUMÉ
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of "hinge" proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled.
Sujet(s)
Ingénierie des protéines , Cristallographie aux rayons X , Ligands , Ingénierie des protéines/méthodes , Conformation des protéinesRÉSUMÉ
DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). These results increase DSD output bandwidth by an order of magnitude over what is currently feasible with fluorescence spectroscopy.
Sujet(s)
Nanopores , ADN , Séquençage nucléotidique à haut débit/méthodes , Recombinaison génétique , Analyse de séquence d'ADN/méthodesRÉSUMÉ
Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons1, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
Sujet(s)
Systèmes CRISPR-Cas , ADN , Édition de gène , Expression des gènes , Mémorisation et recherche des informations , ARN , Transcription inverse , Systèmes CRISPR-Cas/génétique , ADN/biosynthèse , ADN/génétique , Édition de gène/méthodes , Génome/génétique , Mémorisation et recherche des informations/méthodes , Integrases/métabolisme , Cellules procaryotes/métabolisme , ARN/génétique , Facteurs tempsRÉSUMÉ
Synthetic DNA is a growing alternative to electronic-based technologies in fields such as data storage, product tagging, or signal processing. Its value lies in its characteristic attributes, namely Watson-Crick base pairing, array synthesis, sequencing, toehold displacement and polymerase chain reaction (PCR) capabilities. In this review, we provide an overview of the most prevalent applications of synthetic DNA that could shape the future of information technology. We emphasize the reasons why the biomolecule can be a valuable alternative for conventional electronic-based media, and give insights on where the DNA-analog technology stands with respect to its electronic counterparts.
Sujet(s)
ADN/composition chimique , Technologie de l'information ,RÉSUMÉ
Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.
Sujet(s)
Nanopores , Bactéries , HumainsRÉSUMÉ
Proteins carry out life's essential functions. Comprehensive proteome analysis technologies are thus required for a full understanding of the operating principles of biological systems. While current proteomics techniques suffer from limitations in sensitivity and/or throughput, nanopore technology has the potential to enable de novo protein identification through single-molecule sequencing. However, a significant barrier to achieving this goal is controlling protein/peptide translocation through the nanopore sensor for processive strand analysis. Here, we review recent approaches that use a range of techniques, from oligonucleotide conjugation to molecular motors, aimed at driving protein strands and peptides through protein nanopores. We further discuss site-specific protein conjugation chemistry that could be combined with these translocation approaches as future directions to achieve single-molecule protein detection and sequencing of native proteins.
RÉSUMÉ
DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of â¼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.
Sujet(s)
Nanopores , ADN , DNA-directed DNA polymerase , Cinétique , Analyse de séquence d'ADNRÉSUMÉ
Proteins present a significant challenge for nanopore-based sequence analysis. This is partly due to their stable tertiary structures that must be unfolded for linear translocation, and the absence of regular charge density. To address these challenges, here we describe how ClpXP, an ATP-dependent protein unfoldase, can be harnessed to unfold and processively translocate multi-domain protein substrates through an alpha-hemolysin nanopore sensor. This process results in ionic current patterns that are diagnostic of protein sequence and structure at the single-molecule level.
Sujet(s)
Endopeptidase Clp/métabolisme , Hémolysines/composition chimique , Hémolysines/métabolisme , Double couche lipidique/métabolisme , Nanopores , Dépliement des protéines , Transport des protéinesRÉSUMÉ
Molecular tagging is an approach to labeling physical objects using DNA or other molecules that can be used when methods such as RFID tags and QR codes are unsuitable. No molecular tagging method exists that is inexpensive, fast and reliable to decode, and usable in minimal resource environments to create or read tags. To address this, we present Porcupine, an end-user molecular tagging system featuring DNA-based tags readable within seconds using a portable nanopore device. Porcupine's digital bits are represented by the presence or absence of distinct DNA strands, called molecular bits (molbits). We classify molbits directly from raw nanopore signal, avoiding basecalling. To extend shelf life, decrease readout time, and make tags robust to environmental contamination, molbits are prepared for readout during tag assembly and can be stabilized by dehydration. The result is an extensible, real-time, high accuracy tagging system that includes an approach to developing highly separable barcodes.
Sujet(s)
ADN/génétique , Nanopores , Biologie synthétique/méthodes , Algorithmes , Biologie informatique , Systèmes informatiques , Traitement automatique des données , Apprentissage machine , Analyse de séquence d'ADNRÉSUMÉ
Molecular data storage is an attractive alternative for dense and durable information storage, which is sorely needed to deal with the growing gap between information production and the ability to store data. DNA is a clear example of effective archival data storage in molecular form. In this Review, we provide an overview of the process, the state of the art in this area and challenges for mainstream adoption. We also survey the field of in vivo molecular memory systems that record and store information within the DNA of living cells, which, together with in vitro DNA data storage, lie at the growing intersection of computer systems and biotechnology.
Sujet(s)
ADN/génétique , Mémorisation et recherche des informations/méthodes , Animaux , Biotechnologie/méthodes , HumainsRÉSUMÉ
The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.
Sujet(s)
Systèmes CRISPR-Cas/génétique , Clustered regularly interspaced short palindromic repeats , ADN intergénique/génétique , Locus génétiques , Escherichia coli/génétique , Évolution moléculaire , Génome bactérien , Sulfolobus/génétique , Yersinia pestis/génétiqueRÉSUMÉ
DNA is an excellent medium for archiving data. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity. When harnessed, this system has the potential to write arbitrary information into the genome. Here we use the CRISPR-Cas system to encode the pixel values of black and white images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We also uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition that are relevant for understanding both the basic biology of bacterial adaptation and its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells.