Scalable Combinatorial Assembly of Synthetic DNA for Tracking Applications.

Synthetic DNA barcodes are double-stranded DNA molecules designed to carry recoverable information, information that can be used to represent and track objects and organisms. DNA barcodes offer robust, sensitive detection using standard amplification and sequencing techniques. While numerous research groups have promoted DNA as an information storage medium, less attention has been devoted to the design of economical, scalable DNA barcode libraries. Here, we present an alternative modular approach to sequence design. Barcode sequences were constructed from smaller, interchangeable blocks, allowing for the combinatorial assembly of numerous distinct tags. We demonstrated the design and construction of first-generation (N = 256) and second-generation (N = 512) modular barcode libraries, from fewer than 50 total single-stranded oligonucleotides for each library. To avoid contamination during experimental validation, a liquid-handling robot was employed for oligonucleotide mixing. Generating barcode sequences in-house reduces dependency upon external entities for unique tag generation, increasing flexibility in barcode generation and deployment. Next generation sequencing (NGS) detection of 256 different samples in parallel highlights the multiplexing afforded by the modular barcode design coupled with high-throughput sequencing. Deletion variant analysis of the first-generation library informed sequence design for enhancing barcode assembly specificity in the second-generation library.

Lanthanide (Eu3+/Tb3+)-Loaded γ-Cyclodextrin Nano-Aggregates for Smart Sensing of the Anticancer Drug Irinotecan.

The clinical use of anticancer drugs necessitates new technologies for their safe, sensitive, and selective detection. In this article, lanthanide (Eu3+ and Tb3+)-loaded γ-cyclodextrin nano-aggregates (ECA and TCA) are reported, which sensitively detects the anticancer drug irinotecan by fluorescence intensity changes. Fluorescent lanthanide (Eu3+ and Tb3+) complexes exhibit high fluorescence intensity, narrow and distinct emission bands, long fluorescence lifetime, and insensitivity to photobleaching. However, these lanthanide (Eu3+ and Tb3+) complexes are essentially hydrophobic, toxic, and non-biocompatible. Lanthanide (Eu3+ and Tb3+) complexes were loaded into naturally hydrophilic γ-cyclodextrin to form fluorescent nano-aggregates. The biological nontoxicity and cytocompatibility of ECA and TCA fluorescent nanoparticles were demonstrated by cytotoxicity experiments. The ECA and TCA fluorescence nanosensors can detect irinotecan selectively and sensitively through the change of fluorescence intensity, with detection limits of 6.80 µM and 2.89 µM, respectively. ECA can safely detect irinotecan in the cellular environment, while TCA can detect irinotecan intracellularly and is suitable for cell labeling.

Advancing biomarkers for anaerobic o-xylene biodegradation via metagenomic analysis of a methanogenic consortium.

Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.

Installing Guest Molecules at Specific Sites within Scaffold Protein Crystals.

Protein crystals are porous self-assembling materials that can be rapidly evolved by mutagenesis. We aimed to develop scaffold assisted crystallography techniques in an engineered protein crystal with large pores (>13 nm). Guest molecules were installed via a single covalent bond to attempt to reduce the conformational freedom and achieve high-occupancy structures. We used four different conjugation strategies to attach guest molecules to three different cysteine sites within pre-existing protein crystals. In all but one case, the presence of the adduct was obvious in the electron density. Structure determination of larger guest molecules may be feasible due to the large pores of the engineered scaffold crystals.

Programmed Assembly of Host-Guest Protein Crystals.

The binding and release of guest fluorescent proteins inside a protein crystal with 13 nm axial pores is controlled. Spatially segregated guest protein loading is achieved via sequential binding and release stages. Additionally, selective stabilization of the crystal exterior results in hollow crystalline shells.

An Evolved Orthogonal Enzyme/Cofactor Pair.

We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

Characterization of the target of ivermectin, the glutamate-gated chloride channel, from Anopheles gambiae.

The use of insecticide-treated nets and indoor residual insecticides targeting adult mosquito vectors is a key element in malaria control programs. However, mosquito resistance to the insecticides used in these applications threatens malaria control efforts. Recently, the mass drug administration of ivermectin (IVM) has been shown to kill Anopheles gambiae mosquitoes and disrupt Plasmodium falciparum transmission in the field. We cloned the molecular target of IVM from A. gambiae, the glutamate-gated chloride channel (AgGluCl), and characterized its transcriptional patterns, protein expression and functional responses to glutamate and IVM. AgGluCl cloning revealed an unpredicted fourth splice isoform as well as a novel exon and splice site. The predicted gene products contained heterogeneity in the N-terminal extracellular domain and the intracellular loop region. Responses to glutamate and IVM were measured using two-electrode voltage clamp on Xenopus laevis oocytes expressing AgGluCl. IVM induced non-persistent currents in AgGluCl-a1 and did not potentiate glutamate responses. In contrast, AgGluCl-b was insensitive to IVM, suggesting that the AgGluCl gene could produce IVM-sensitive and -insensitive homomultimers from alternative splicing. AgGluCl isoform-specific transcripts were measured across tissues, ages, blood feeding status and sex, and were found to be differentially transcribed across these physiological variables. Lastly, we stained adult, female A. gambiae for GluCl expression. The channel was expressed in the antenna, Johnston's organ, supraesophageal ganglion and thoracic ganglia. In summary, we have characterized the first GluCl from a mosquito, A. gambiae, and described its unique activity and expression with respect to it as the target of the insecticide IVM.

Porous protein crystals: synthesis and applications.

Large-pore protein crystals (LPCs) are an emerging class of biomaterials. The inherent diversity of proteins translates to a diversity of crystal lattice structures, many of which display large pores and solvent channels. These pores can, in turn, be functionalized via directed evolution and rational redesign based on the known crystal structures. LPCs possess extremely high solvent content, as well as extremely high surface area to volume ratios. Because of these characteristics, LPCs continue to be explored in diverse applications including catalysis, targeted therapeutic delivery, templating of nanostructures, structural biology. This Feature review article will describe several of the existing platforms in detail, with particular focus on LPC synthesis approaches and reported applications.

PAbFold: Linear Antibody Epitope Prediction using AlphaFold2.

Defining the binding epitopes of antibodies is essential for understanding how they bind to their antigens and perform their molecular functions. However, while determining linear epitopes of monoclonal antibodies can be accomplished utilizing well-established empirical procedures, these approaches are generally labor- and time-intensive and costly. To take advantage of the recent advances in protein structure prediction algorithms available to the scientific community, we developed a calculation pipeline based on the localColabFold implementation of AlphaFold2 that can predict linear antibody epitopes by predicting the structure of the complex between antibody heavy and light chains and target peptide sequences derived from antigens. We found that this AlphaFold2 pipeline, which we call PAbFold, was able to accurately flag known epitope sequences for several well-known antibody targets (HA / Myc) when the target sequence was broken into small overlapping linear peptides and antibody complementarity determining regions (CDRs) were grafted onto several different antibody framework regions in the single-chain antibody fragment (scFv) format. To determine if this pipeline was able to identify the epitope of a novel antibody with no structural information publicly available, we determined the epitope of a novel anti-SARS-CoV-2 nucleocapsid targeted antibody using our method and then experimentally validated our computational results using peptide competition ELISA assays. These results indicate that the AlphaFold2-based PAbFold pipeline we developed is capable of accurately identifying linear antibody epitopes in a short time using just antibody and target protein sequences. This emergent capability of the method is sensitive to methodological details such as peptide length, AlphaFold2 neural network versions, and multiple-sequence alignment database. PAbFold is available at https://github.com/jbderoo/PAbFold.

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Šof the enzyme active site. This mutation places a second H residue within 5 Šof an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG, and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained the ability to reduce SeO42-. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

Modular Protein-DNA Cocrystals as Precise, Programmable Assembly Scaffolds.

High-precision nanomaterials to entrap DNA-binding molecules are sought after for applications such as controlled drug delivery and scaffold-assisted structural biology. Here, we engineered protein-DNA cocrystals to serve as scaffolds for DNA-binding molecules. The designed cocrystals, isoreticular cocrystals, contain DNA-binding protein and cognate DNA blocks where the DNA-DNA junctions stack end-to-end. Furthermore, the crystal symmetry allows topology preserving (isoreticular) expansion of the DNA stack without breaking protein-protein contacts, hence providing larger solvent channels for guest diffusion. Experimentally, the resulting designed isoreticular cocrystal adopted an interpenetrating I222 lattice, a phenomenon previously observed in metal-organic frameworks (MOFs). The interpenetrating lattice crystallized dependably in the same space group despite myriad modifications at the DNA-DNA junctions. Assembly was modular with respect to the DNA inserted for expansion, providing an interchangeable DNA sequence for guest-specified scaffolding. Also, the DNA-DNA junctions were tunable, accommodating varied sticky base overhang lengths and terminal phosphorylation. As a proof of concept, we used the interpenetrating scaffold crystals to separately entrap three distinct guest molecules during crystallization. Isoreticular cocrystal design offers a route to a programmable scaffold for DNA-binding molecules, and the design principles may be applied to existing cocrystals to develop scaffolding materials.

Ligand Presentation Inside Protein Crystal Nanopores: Tunable Interfacial Adhesion Noncovalently Modulates Cell Attachment.

Protein crystals with sufficiently large solvent pores can non-covalently adsorb polymers in the pores. In principle, if these polymers contain cell adhesion ligands, the polymer-laden crystals could present ligands to cells with tunable adhesion strength. Moreover, porous protein crystals can store an internal ligand reservoir, so that the surface can be replenished. In this study, we demonstrate that poly(ethylene glycol) terminated with a cyclic cell adhesion ligand peptide (PEG-RGD) can be loaded into porous protein crystals by diffusion. Through atomic force microscopy (AFM), force-distance correlations of the mechanical interactions between activated AFM tips and protein crystals were precisely measured. The activation of AFM tips allows the tips to interact with PEG-RGD that was pre-loaded in the protein crystal nanopores, mimicking how a cell might attach to and pull on the ligand through integrin receptors. The AFM experiments also simultaneously reveal the detailed morphology of the buffer-immersed nanoporous protein crystal surface. We also show that porous protein crystals (without and with loaded PEG-RGD) serve as suitable substrates for attachment and spreading of adipose-derived stem cells. This strategy can be used to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.

A family of thermostable fungal cellulases created by structure-guided recombination.

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63 degrees C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63 degrees C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 degrees C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.

Conditionally designed luminescent DNA crystals doped by Ln3+(Eu3+/Tb3+) complexes or fluorescent proteins with smart drug sensing property.

In this work, a designed porous DNA crystal with high intrinsic biocompatibility was used as the scaffold material to load fluorescent guest molecules to detect anti-cancer drugs. It is shown here that the synthesized crystals have the characteristics consistent with the designed large solvent channels, and can therefore accommodate guest molecules such as fluorescent proteins that cannot be accommodated by less porous crystals. Eu(TTA)3phen and Tb(acac)3phen lanthanide complexes were individually noncovalently loaded into the porous crystals, resulting in hybrid luminescent DNA crystals. Emodin, an anti-cancer, anti-tumor, anti-inflammatory drug, was found to quench lanthanide complexes in solution or in crystals. Notably, emodin is the active ingredient of Lianhua Qingwen Capsule, an anti-COVID-19 drug candidate. Therefore, the porous DNA crystals reported here have potential applications as a biocompatible and theranostic delivery biomaterial for functional macromolecules.

Mosquito tagging using DNA-barcoded nanoporous protein microcrystals.

Conventional mosquito marking technology for mark-release-recapture (MRR) is quite limited in terms of information capacity and efficacy. To overcome both challenges, we have engineered, lab-tested, and field-evaluated a new class of marker particles, in which synthetic, short DNA oligonucleotides (DNA barcodes) are adsorbed and protected within tough, crosslinked porous protein microcrystals. Mosquitoes self-mark through ingestion of microcrystals in their larval habitat. Barcoded microcrystals persist trans-stadially through mosquito development if ingested by larvae, do not significantly affect adult mosquito survivorship, and individual barcoded mosquitoes are detectable in pools of up to at least 20 mosquitoes. We have also demonstrated crystal persistence following adult mosquito ingestion. Barcode sequences can be recovered by qPCR and next-generation sequencing (NGS) without detectable amplification of native mosquito DNA. These DNA-laden protein microcrystals have the potential to radically increase the amount of information obtained from future MRR studies compared to previous studies employing conventional mosquito marking materials.

Polarizable protein packing.

To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol(-1)] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems.

Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

Non-bulk-like solvent behavior in the ribosome exit tunnel.

As nascent proteins are synthesized by the ribosome, they depart via an exit tunnel running through the center of the large subunit. The exit tunnel likely plays an important part in various aspects of translation. Although water plays a key role in many bio-molecular processes, the nature of water confined to the exit tunnel has remained unknown. Furthermore, solvent in biological cavities has traditionally been characterized as either a continuous dielectric fluid, or a discrete tightly bound molecule. Using atomistic molecular dynamics simulations, we predict that the thermodynamic and kinetic properties of water confined within the ribosome exit tunnel are quite different from this simple two-state model. We find that the tunnel creates a complex microenvironment for the solvent resulting in perturbed rotational dynamics and heterogenous dielectric behavior. This gives rise to a very rugged solvation landscape and significantly retarded solvent diffusion. We discuss how this non-bulk-like solvent is likely to affect important biophysical processes such as sequence dependent stalling, co-translational folding, and antibiotic binding. We conclude with a discussion of the general applicability of these results to other biological cavities.