Recombinant adeno-associated viruses (AAVs) with precise genome editing and cell-virus interaction have become a promising delivery tool for gene therapy. A robust AAV purification process is crucial for ensuring therapeutic efficacy. The challenges of AAV purification process development encompass limited material availability during early-stage development, high cost-of-goods compared to traditional biologics, and short development timelines for the critical first-in-human stages. The key to overcoming these challenges is to leverage high throughput (HTP) methods. In this article, an integrated end-to-end HTP workflow is proposed, utilizing a resin tip as the purification module and incorporating an HTP analytical toolkit on one platform. Purification parameters, including binding capacity, resin selection, and buffer composition screening for AAV full/partial/empty capsids separation, are efficiently determined using a 25 μL resin tip and HTP analytical tools with only micro-volume sample requirements. The process parameters determined from the HTP workflow predict the trends of full capsid enrichment and partial capsid removal for the bench-scale purification. This HTP workflow is also applied for the assessment of the AAV quality attributes to accelerate early-stage cell line and cell culture development. Comparable AAV quality attributes are demonstrated to Robocolumn as the benchmark HTP purification method. By leveraging HTP analytical tools to instantly interpret the purification data, this integrated HTP workflow effectively accelerates AAV purification process development, with a 2% material volume requirement compared to the benchmark method, 96-well format screening, short turnaround time for analytical assays, and significant cost-of-goods savings for downstream process development.

ALternative Isoinformational ENgineered” (ALIEN) DNA is a biomimetic polymer composed of four entirely anthropogenic nucleotides. These alternative nucleosides form base pairs orthogonal to canonical bases and fold into the familiar B-form DNA double-helix, endowing ALIEN DNA with valuable biotechnological applications. The ability to sequence ALIEN DNA is essential for its continued development. However traditional sequencing approaches rely on chemical recognition of ACGT-DNA and cannot be easily adapted to ALIEN DNA. Here we demonstrate de novo nanopore sequencing of DNA comprised entirely of the four anthropogenic DNA bases. We show direct, label-free, single-molecule sequencing of such nucleic acids without the requirements of fluorescent labels, transliteration, amplification, or enzymatic synthesis. This paves the way for routine, accessible, and high-accuracy sequencing of DNA beyond A, C, G, and T.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

This Perspective addresses the unresolved, and still hotly contested, question of how enzymes transition from stable enzyme-substrate (ES) complexes to successful, femtosecond barrier crossings. By extending Marcus theory to enzyme-catalyzed reactions, we argue that environmental reorganization of the protein scaffold, together with associated water molecules, achieves the intersection of reactant and product potential energy surfaces. After discussing the experimentally demonstrated importance of reduced activation enthalpy in enzyme-catalyzed transformations, we describe new methodologies that measure the temperature dependence of (i) time-averaged hydrogen/deuterium exchange into backbone amides and (ii) time-dependent Stokes shifts to longer emission wavelengths in appended chromophores at the protein/water interface. These methods not only identify specific pathways for the transfer of thermal energy from solvent to the reacting bonds of bound substrates but also suggest that collective thermally activated protein restructuring must occur very rapidly (on the ns–ps time scale) over long distances. Based on these findings, we introduce a comprehensive model for how barrier crossing takes place from the ES complex. This exploits the structural preorganization inherent in protein folding and subsequent conformational sampling, which optimally positions essential catalytic components within ES ground states and correctly places reactive bonds in the substrate(s) relative to embedded energy transfer networks connecting the protein surface to the active site. The existence of these anisotropic energy distribution pathways introduces a new dimension into the ongoing quest for improved de novo enzyme design.

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called 'unnatural' nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering.

Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2n daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. tny1 mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. TNY1 encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of TNY1 in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed TNY1 function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in tny1 cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.

Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest “cargo” are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.

All known bacterial tRNAs adopt the canonical cloverleaf 2D and L-shaped 3D structures. We aimed to explore whether alternative tRNA structures could be introduced in bacterial translation. To this end, we crafted a vitamin-based genetic system to evolve Escherichia coli toward activity of structurally non-canonical tRNAs. The system reliably couples (escape frequency <10−12) growth with the activities of a novel orthogonal histidine suppressor tRNA (HisTUAC) and of the cognate ARS (HisS) via suppression of a GTA valine codon in the mRNA of an enzyme in thiamine biosynthesis (ThiN). Suppression results in the introduction of an essential histidine and thereby confers thiamine prototrophy. We then replaced HisTUAC in the system with non-canonical suppressor tRNAs and selected for growth. A strain evolved to utilize mini HisT, a tRNA lacking the D-arm, and we identified the responsible mutation in an RNase gene (pnp) involved in tRNA degradation. This indicated that HisS, the ribosome, and EF-Tu accept mini HisT ab initio, which we confirmed genetically and through in vitro translation experiments. Our results reveal a previously unknown flexibility of the bacterial translation machinery for the accepted fold of the adaptor of the genetic code and demonstrate the power of the vitamin-based suppression system.

Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system in vivo and showcase its functionality by importing an impermeant non-canonical amino acid.

By rearranging hydrogen bond donor and acceptor groups within a standard Watson–Crick geometry, DNA can add eight independently replicable nucleotides forming four additional not found in standard Terran DNA. For many applications, the orthogonal pairing of standard and nonstandard pairs offers a key advantage. However, other applications require standard and nonstandard nucleotides to communicate with each other. This is especially true when seeking to recruit high-throughput instruments (e.g., Illumina), designed to sequence standard 4-nucleotide DNA, to sequence DNA that includes added nucleotides. For this purpose, PCR workflows are needed to replace nonstandard nucleotides in (for example) a 6-letter DNA sequence by defined mixtures of standard nucleotides built from 4 nucleotides. High-throughput sequencing can then report the sequences of those mixtures to bioinformatic alignment tools, which infer the original 6-nucleotide sequence by analysis of the mixtures. Unfortunately, the intrinsic orthogonality of standard and nonstandard nucleotides often demand polymerases that violate pairing biophysics to do this replacement, leading to inefficiencies in this “transliteration” process. Thus, laboratory in vitro evolution (LIVE) using “anthropogenic evolvable genetic information systems” (AEGIS), an important “consumer” of new sequencing tools, has been slow to be democratized; robust sequencing is needed to identify the AegisBodies and AegisZymes that AEGIS-LIVE delivers. This work introduces a new way to connect synthetic and standard molecular biology: biversal nucleotides. In an example presented here, a pyrimidine analogue (pyridine-2-one, y) pairs with Watson–Crick geometry to both a nonstandard base (2-amino-8-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one, P, the Watson–Crick partner of 6-amino-5-nitro-[1H]-pyridin-2-one, Z) and a base that completes the Watson–Crick hydrogen bond pattern (2-amino-2'-deoxyadenosine, amA). PCR amplification of GACTZP DNA with dyTP delivers products where Z:P pairs are cleanly transliterated to A:T pairs. In parallel, PCR of the same GACTZP sample at higher pH delivers products where Z:P pairs are cleanly transliterated to C:G pairs. By allowing robust sequencing of 6-letter GACTZP DNA, this workflow will help democratize AEGIS-LIVE. Further, other implementations of the biversal concept can enable communication across and between standard DNA and synthetic DNA more generally.

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Arginine phosphorylation plays numerous roles throughout biology. Arginine kinase (AK) catalyzes the delivery of an anionic phosphoryl group (PO₃^–) from ATP to a planar, trigonal nitrogen in a guanidinium cation. Density functional theory (DFT) calculations have yielded a model of the transition state (TS) for the AK-catalyzed reaction. They reveal a network of over 50 hydrogen bonds that delivers unprecedented pyramidalization and out-of-plane polarization of the arginine guanidinium nitrogen (Nη2) and aligns the electron density on Nη2 with the scissile P–O bond, leading to in-line phosphoryl transfer via an associative mechanism. In the reverse reaction, the hydrogen-bonding network enforces the conformational distortion of a bound phosphoarginine substrate to increase the basicity of Nη2. This enables Nη2 protonation, which triggers PO₃^– migration to generate ATP. This polarization–pyramidalization of nitrogen in the arginine side chain is likely a general phenomenon that is exploited by many classes of enzymes mediating the post-translational modification of arginine.