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Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo–C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecBexo–C were thoroughly explored using 0.03–25 kb dsDNAs. When combined with RecBexo–C, CbAgo could cleave targets located 11–12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecBexo–C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecBexo–C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.

The formation and persistence of DNA damage can impact biological processes such as DNA replication and transcription. To maintain genome stability and integrity, organisms rely on robust DNA damage repair pathways. Techniques to detect and locate DNA damage sites across a genome enable an understanding of the consequences of DNA damage as well as how damage is repaired, which can have key diagnostic and therapeutic implications. Importantly, advancements in technology have enabled the development of high-throughput sequencing-based DNA damage detection methods. These methods require DNA enrichment or amplification steps that limit the ability to quantitate the DNA damage sites. Further, each of these methods is typically tailored to detect only a specific type of damage. RAre DAmage and Repair (RADAR) sequencing is a DNA sequencing workflow that overcomes these limitations and enables detection and quantitation of DNA damage sites in any organism on a genome-wide scale. RADAR-seq works by replacing DNA damage sites with a patch of modified bases that can be directly detected by Pacific Biosciences Single-Molecule Real Time sequencing. Here, we present three protocols that enable detection of thymine dimers and ribonucleotides in bacterial and archaeal genomes. Basic Protocol 1 enables construction of a reference genome required for RADAR-seq analyses. Basic Protocol 2 describes how to locate, quantitate, and compare thymine dimer levels in Escherichia coli exposed to varying amounts of UV light. Basic Protocol 3 describes how to locate, quantitate, and compare ribonucleotide levels in wild-type and ΔRNaseH2 Thermococcus kodakarensis. Importantly, all three protocols provide in-depth steps for data analysis. Together they serve as proof-of-principle experiments that will allow users to adapt the protocols to locate and quantitate a wide variety of DNA damage sites in any organism. © 2022 New England Biolabs. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Constructing a reference genome utilizing SMRT sequencing Basic Protocol 2: Mapping and quantitating genomic thymine dimer formation in untreated versus UV-irradiated E. coli using RADAR-seq Basic Protocol 3: Mapping and quantitating genomic ribonucleotide incorporation in wildtype versus ΔRNaseH2 T. kodakarensis using RADAR-seq.

We develop a connection between DP-colorings of $k$-uniform hypergraphs of order $n$ and coverings of $n$-dimensional Boolean hypercube by pairs of antipodal $(n-k)$-dimensional faces. Bernshteyn and Kostochka established a lower bound on the number of edges in a non-2-DP-colorable $k$-uniform hypergraph namely, $2^{k-1}$ for odd $k$ and $2^{k-1}+1$ for even $k.$ They proved that these bounds are tight for $k=3,4$. In this paper, we prove that the bound is achieved for all odd $k\geq 3$.

In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs—such as pseudouridine (Ψ) and N1-methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro.

In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs-such as pseudouridine (Ψ) and N

The paper is focused on evaluating the oral cavity homeostasis in patients with mild chronic generalized periodontitis (CGP) (Group 1, 35 persons) and mild peri-implantitis (Group 2, 30 persons). The control group included healthy individuals (20 persons) with neither dental nor somatic issues. The material used for the study was oral fluid. The general metabolic processes in the periodontal and peri-implant area tissues were described based on analysis of the protein and mineral metabolism indicators, as well as on the hygiene and periodontal status indices. The paper presents the results of a comparative assessment involving Groups I and II with the control group and between Groups I and II. Our findings show changes in ion balance in both groups. Significant disturbances in the protein and mineral metabolism have been identified in the main groups, which contributes to the development of inflammation and reduced detoxification in periodontal and peri-implant tissues. A rise in Calcium and Magnesium indicates destructive processes in bone tissues. In case of mild CGP, these changes are more prominent. The oral hygiene and periodontal status indices in Group I proved to be significantly worse. Therefore, while performing implantation on the background of chronic generalized periodontitis, it is important to take into account its metabolic imbalance and implement preventive measures

Based on currently available literature, clinical examination remains the major method when handling cases of suspected malignancy. However, this method does not allow diagnosing cancer, due to which a large group of patients with possible oral mucosa cancer are referred to an oncologist. The search and use of affordable non-invasive methods for early diagnosis of oral mucosa tumors is an urgent issue facing the health system. The study involved analyzing 134 records of outpatients examined at the Samara Regional Oncological Clinic who were referred by dentists within 2014-2019 from the local polyclinic in Samara due to detection of tumors in oral mucosa and who underwent a biopsy. The patients were divided into two groups according to the examination methods. The inclusion criteria were: detection of various superficial oral mucosa neoplasms; referral from the dentist. The exclusion criteria were as follows: patients with submucosal oral cavity neoplasms referred to the oncologist by other medical specialists or self-referred patients. The control group included 63 patients who, after a conventional examination (including interview, examination, palpation), underwent an incisional biopsy followed by morphological examination at the oncologist’s office. In the major group, in 71 patients at their respective initial dental appointments a special examination algorithm was applied. This algorithm entailed an assessment of the identified risk factors. Indications for biopsy were identified using the histological verification index (HVI). Apart from the conventional examination methods (interview, examination, palpation), autofluorescence stomatoscopy was used, this being done for the purpose of differential diagnostics of inflammation, precancerous and malignant issues, depending on the glow type. In the main group, the initial stages of oral mucosa cancer were detected in 17 patients after biopsy; in the control group – in 4 patients (p=0.004). The developed algorithm used for scoring the patient’s clinical examination data combined with autofluorescence stomatoscopy allowed diagnosing accurately (90% of reliability) precancerous and cancerous diseases, as well as to use invasive research methods (biopsy) strictly following the indications. Aim of study: to improve diagnosis of oral mucosa neoplasms through improvement of the examination algorithm.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3′-OH and 5′-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

A number of highly efficient methods for the preparation of novel derivatives of 9-selenabicyclo[3.3.1]nonane in high yields based on selenium dibromide and cis,cis-1,5-cyclooctadiene are reported. The one-pot syntheses of 2,6-diorganyloxy-9-selenabicyclo[3.3.1]nonanes using various O-nucleophiles including alkanols, phenols, benzyl, allyl, and propargyl alcohols were developed. New 2,6-bis(1,2,3-triazol-1-yl)-9-selenabicyclo[3.3.1]nonanes were obtained by the copper-catalyzed 1,3-dipolar cycloaddition of 2,6-diazido-9-selenabicyclo[3.3.1]nonane with unsubstituted gaseous acetylene and propargyl alcohol. The synthesis of 2,6-bis(vinylsulfanyl)-9-selenabicyclo[3.3.1]nonane, based on the generation of corresponding dithiolate anion from bis[amino(iminio)methylsulfanyl]-9-selenabicyclo[3.3.1]nonane dibromide, followed by the nucleophilic addition of the dithiolate anion to unsubstituted acetylene, was developed. The glutathione peroxidase-like activity of the obtained water-soluble products was estimated and compounds with high activity were found. Overall, 2,6-Diazido-9-selenabicyclo[3.3.1]nonane exhibits the highest activity among the obtained compounds.

An embedding of a code is a mapping that preserves distances between codewords. We prove that any code with code distance $\rho$ and length $d$ can be embedded into an MDS code with the same code distance and length but under a larger alphabet. As a corollary we obtain embeddings of systems of partial mutually orthogonal Latin cubes and $n$-ary quasigroups., Comment: 7 pages

The catalytic regioselective and highly efficient synthesis of bis-1,2,3-triazole derivatives of 9-selenabicyclo[3.3.1]nonane was developed. The 1,3-dipolar cycloaddition reaction of 2,6-diazido-9-selenabicyclo[3.3.1]nonane with a variety of terminal acetylenes catalyzed by a copper acetate/sodium ascorbate system proceeded in a regioselective fashion, affording 2,6-bis(4-organyl-1,2,3-triazole)-9-selenabicyclo[3.3.1]nonanes in high yields (93–98%). The reaction of 2,6-diazido-9-selenabicyclo[3.3.1]nonane with dimethyl and diethyl acetylenedicarboxylates was carried out as thermal 1,3-dipolar Huisgen cycloaddition giving the corresponding 4,5-disubstituted 1,2,3-triazole derivatives of 9-selenabicyclo[3.3.1]nonane in high yields. The obtained products are potentially bioactive compounds and first representatives of selenium heterocycles combined with two 1,2,3-triazole moieties. 2.6-Diazido-9-selenabicyclo[3.3.1]nonane was obtained in quantitative yield via the reaction of sodium azide with 2,6-dibromo-9-selenabicyclo[3.3.1]nonane at room temperature. The latter compound was synthesized by stereoselective transannular addition of selenium dibromide to cis, cis-1,5-cyclooctadiene.

The first organic metals were obtained based on tetrathiafulvalene. The most significant advance in the field of organic metals was the discovery of superconductivity. The first organic superconductors were obtained based on tetramethyltetraselenafulvalene. These facts demonstrate great importance of tetraselenafulvalenes and their precursors, diselenafulvenes, for materials sciences. Derivatives of 1,4-diselenafulvene and 1,4,5,8-tetraselenafulvalene are useful building blocks for organic synthesis and donor units for the preparation of charge-transfer complexes and radical ion salts, the construction of organic metals, superconductors, organic Dirac materials, semiconductors, ferromagnets, and other conductive materials. This review covers the literature on the design, synthesis, and application of 1,4,5,8-tetraselenafulvalenes and 1,4-diselenafulvenes and their tellurium analogs over the past 15–20 years. These two classes of compounds are interconnected, since the main part of methods for the synthesis of tetraselenafulvalenes is based on the diselenafulvene derivatives as starting compounds. Special attention is paid to the development of novel efficient synthetic approaches to these classes of compounds. Conducting properties and distinguishing features of materials based on tetraselenafulvalenes and their tellurium analogs as well as examples of materials with high conductivity are discussed.

The first results of magnetotelluric studies carried out on the profile of v. Talmenka – Leninsk-Kuznetsky (South-Eastern part of the West Siberian plate and Salair) are considered, the main features of the distribution of deep electrical conductivity in the two main geological structures of the study area: the South-Eastern part of the West Siberian plate and the Salair zone are shown.

The BLL lectin from the edible Japanese “Kurokawa” mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen–Friedenreich antigen (TF-antigen; Galβ1,3GalNAc-α-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and β-GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL’s two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal β-GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

This paper presents the results of magnetotelluric (MT) studies performed within the Western Transbaikalia segment of the Central Asian fold belt along the Selenga River delta-Krasnyi Chikoi Village profile. The data are interpreted using the results of recent geological, petrological, geothermal, tectonic, and geochronological studies of the Mongolia-Transbaikalian part of the Central Asian fold belt, including large heterochronous provinces and zones. The studies have shown that the investigated area has a complex geologic and tectonic structure produced by extensive rifting leading to the formation of large crustal blocks and by intense magmatic fluid activity along deep fault zones. The investigated by a combination of blocks with different types of geoelectric section – mountain range and intermontane basins separated by long-lived deep fault zones. Significantly less tectonic activity of the West Transbaikalian rift zone, compared with the Baikal region of modern rift formation, was reflected in the contrast of the resistivity distribution and heat flow values in the North-Western and South-Eastern parts of the studied profile.

Large DNA constructs (>10 kb), including small genomes and artificial chromosomes, are invaluable tools for genetic engineering and vaccine development. However, the manufacture of these constructs is laborious. To address this problem, we applied new design insights and modified protocols to Golden Gate assembly. While this methodology is routinely used to assemble 5-10 DNA parts in one-step, we found that optimized assembly permitted >50 DNA fragments to be faithfully assembled in a single reaction. We applied these insights to genome construction, carrying out rapid assembly of the 40 kb T7 bacteriophage genome from 52 parts and recovering infectious phage particles after cellular transformation. The new Golden Gate assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets.

Objective of the study: to develop and assess the occlusal digital splint for treating patients with temporomandibular joint dysfunction (TMJD). Material and Methods — 17 patients between the ages of 30 and 49 diagnosed with TMJD were admitted for treatment. To normalize mandibular position, an occlusal digital splint for all patients was manufactured in accordance with our original method. An intensity of pain sensations in all patients prior to, and after, the treatment was assessed by a visual analogue scale, along with the changes in the mandibular movements’ amplitude, and the signs of splint wear and stability of occlusal contacts. Results — Patients have adapted themselves well to the splint and found it convenient in use. The study results showed that it was sufficiently effective in treatment of TMJD. Reduction in pain intensity and restoration of mandibular movements’ amplitude were detected with certainty. Only in 2 (11.7%) cases, the signs of wear on the splint were found, as evidenced by the changes in pre-treatment occlusal contacts. In all other cases throughout the study, a stable occlusal contact has been encountered. Conclusion — Our results indicated that proposed occlusal digital splint may be considered as a treatment option for the patients with TMJ disorders.

A mathematical model of particles movement of separated material in a friction separator is proposed. It includes equations of their movement at each stage of separation (along a rough inclined plane, on a curvilinear part of a springboard, impact of a particle on the surface of a drum, free motion before leaving the separation zone), and differential equations of particle movement in circulating air flow created by a rotating drum; Each of these phases of motion is described by a system of equations obtained on the basis of basic laws of mechanics. An analysis of a particle motion along aslope plane was carried out on the basis of the law on energy changes. Depending on the ratios of the recovery factor and instantaneous friction, being random variables and also the coordinates of a point impact the particle is possible to move along different trajectories with different initial conditions. The mathematical model of the process of separation of loose multicomponent materials on a friction drum-shelf separator makes it possible to investigate comprehensively the process of separation of particles on friction and elastic properties and to optimize the operating modes of the apparatus at relatively low costs without using macaques and prototypes and investigate them experimentally. On the basis of analysis of the proposed theories of particles motion, the main ways are formulated of further improvement of methodology for designing and creating friction devices.

DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. For example, selection of the DNA sequences at fusion sites between fragments is based on broad assembly guidelines or pre-vetted sets of junctions, rather than being customized for a particular application or cloning project. To facilitate the design of robust assembly reactions, we developed a high-throughput DNA sequencing assay to examine reaction outcomes of Golden Gate assembly with T4 DNA ligase and the most commonly used Type IIS restriction enzymes that generate three-base and four-base overhangs. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. These webtools can be used to create customized assemblies from a target DNA sequence or a desired number of fragments. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. Full implementation of the tools developed here enables direct expansion of existing assembly standards for modular cloning systems (e.g. MoClo) as well as the formation of robust new high-fidelity standards.

There were studied thermophysical properties of meat systems, based on comminuted beef with different chemical compositions in the process of freezing-defrosting. It was demonstrated, that the morphological base of meat (the ratio of muscular, connective and fatty tissues) and its chemical composition essentially influence thermophysical properties in the process of freezing-defrosting. It was revealed, that under conditions of realization of freezing-defrosting cycle with the fat mass share increase, the temperature increase of the maximal speed of crystal creation, decrease of the cryoscopic temperature interval, increase of the share of moisture that changes its aggregate state in the cryoscopic temperature interval, increase of specific heat of the phase transfer in the cryoscopic temperature interval are observed in a meat system. The type of changes of the cryoscopic temperature interval allows to detect that fat essentially influences the process of freezing-defrosting. The realized studies of comparison of thermodynamic changes in meat systems at refrigerator processing allowed to determine the strategy of saving meat systems’ stability that is in providing meat proteins’ colloid stability and renewing initial properties of the meat raw material after storage and defrosting.

Subject. The increasing need for searching of new methods of diagnosis of diseases of a temporal and mandibular joint, is caused by high extent of distribution. The leading experts studying pathology ви - a juicy and mandibular joint, recognize need of studying of driving of a mandible. Purpose. Studying of graphic record of movements of a mandible. Methodology. We carried out registration of the movements of the lower jaw in three mutually perpendicular planes at 122 surveyed aged from 18 up to 25 years, 100 women and 22 men. Within collateral research work of SAMGMU and MSTU (The Moscow State technical university), on the basis of MSTU of «STANKIN» the project on creation of system of registration of the movement of a mandible for diagnosis of diseases ви - a juicy and mandibular joint was realized. The device has two webcams which record and write down the movements of a mandible, by means of keeping track of by the marker attached on the most acting chin point. Results At registration of the movements of a mandible we analyzed the period during 5 serial acts of an unclosing of a mouth. The program transformed the obtained data to schedules (Fig. 4-A, B, C of the application) on which vertical and sagittal deflections during research time are displayed. The possibility of system with a high precision to reflect trajectories of movements and to define at what moment of an unclosing or shutting of a mouth occurs the moment of «failure» — a sharp deviation from the median line. Conclusions. The advantage of the offered method is realization of non-contact technology of registration of the movements of a mandible. In difference from the analog systems of registration of the movements we apply only a measuring marker on a chin which does not obstruct natural traffic of a mandible and does not allow the doctor to influence reliability of received information.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.

A variant of 9°N DNA polymerase [Genbank ID (AAA88769.1)] with three mutations (D141A, E143A, A485L) and commercialized under the name “Therminator DNA polymerase” has the ability to incorporate a variety of modified nucleotide classes. This Review focuses on how Therminator DNA Polymerase has enabled new technologies in synthetic biology and DNA sequencing. In addition, we discuss mechanisms for increased modified nucleotide incorporation.

Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

Modern synthetic biology depends on the manufacture of large DNA constructs from libraries of genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly, facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has typically been accomplished by using small sets of empirically vetted junction pairs, limiting the number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20 fragments in a single reaction.