Ligation Reaction Research Articles

Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes.

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. As one type of disease is always connected to several SNP sites, there is great demand for a reliable multiple SNP detection method. Herein, we mimicked a ligation reaction based on DNA ligase and originally utilized an enzyme-free DNA template-directed click reaction for SNP detection. With 5'-alkyne and 3'-azide groups labelled on two oligonucleotide probes, the target DNA-directed Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction produced a new DNA strand with a triazole backbone, as a mimic of a DNA phosphodiester linkage. Trace amounts of the target (as low as 25 fmol in 50 μL) could be sensitively detected using capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Meanwhile, SNP caused an obvious difference in the efficiency of the click reaction, and 0.5% SNP could be easily detected. More importantly, multiplexed SNP detection in a one tube reaction was successfully achieved only by encoding different lengths of the DNA probes for the different SNP sites.

Peptide nucleic acid-templated selenocystine-selenoester ligation enables rapid miRNA detection.

The development of a rapid and chemoselective selenocystine-selenoester peptide ligation that operates at nanomolar reactant concentrations has been developed by utilising PNA templation. Kinetic analysis of the templated peptide ligation revealed that the selenocystine-selenoester reaction was 10 times faster than traditional native chemical ligation at cysteine and to our knowledge is the fastest templated ligation reaction reported to date. The efficiency and operational simplicity of this technology is highlighted through the formation of hairpin molecular architectures and in a novel paper-based lateral flow assay for the rapid and sequence specific detection of oligonucleotides, including miRNA in cell lysates.

Chemical Ligation Reactions of Oligonucleotides for Biological and Medicinal Applications.

Chemical ligation of oligonucleotides (ONs) is the key reaction for various ON-based technologies. We have tried to solve the problems of RNA interference (RNAi) technology by applying ON chemical ligation to RNAi. We designed a new RNAi system, called intracellular buildup RNAi (IBR-RNAi), where the RNA fragments are built up into active small-interference RNA (siRNA) in cells through a chemical ligation reaction. Using the phosphorothioate and iodoacetyl groups as reactive functional groups for the ligation, we achieved RNAi effects without inducing immune responses. Additionally, we developed a new chemical ligation for IBR-RNAi, which affords a more native-like structure in the ligated product. The new ligation method should be useful not only for IBR-RNAi but also for the chemical synthesis of biofunctional ONs.

A computational model to predict the Diels\u2013Alder reactivity of aryl/alkyl-substituted tetrazines

The tetrazine ligation is one of the fastest bioorthogonal ligations and plays a pivotal role in time-critical in vitro and in vivo applications. However, prediction of the reactivity of tetrazines in inverse electron demand Diels–Alder-initiated ligation reactions is not straight-forward. Commonly used tools such as frontier molecular orbital theory only give qualitative and often even wrong results. Applying density functional theory, we have been able to develop a simple computational method for the prediction of the reactivity of aryl/alkyl-substituted tetrazines in inverse electron demand Diels–Alder reactions.Graphical

Self-primer and self-template recycle rolling circle amplification strategy for sensitive detection of uracil-DNA glycosylase activity

Sensitive and accurate detection of uracil-DNA glycosylase (UDG) activity is available for evaluating and validating their function in uracil base-excision repair (UBER) pathway and clinical diagnosis. Here, a sensitive and accurate method for UDG activity detection was developed on the basis of self-primer and self-template recycle rolling circle amplification (Self-RRCA) strategy. First, an immature template (IT) with a uracil base and an Nt.BbvCI nicking site was designed, which could hybridize with a designed primer to form a pre-amplicon probe (PA probe). Under the action of UDG, the uracil base in the PA probe could be removed to generate an apyrimidinic (AP) site. Then the generated AP site was excised by endonuclease IV (endo IV), making the PA probe form a RCA amplicon through reconformation. The RCA amplicon subsequently was used to trigger the RCA, and after Nt.BbvCI nicking reaction, new amplicons were released to initiate next RCA, constituting a Self-RRCA. In this method, the designed IT was not fully complementary with the primer in the ligation part, which could effectively avoid nonspecific ligation reaction and eventually effectively avoid nonspecific amplification. Compared with the linear RCA, the Self-RRCA exhibited higher amplification efficiency. Due to above advantages, a sensitive and accurate detection method was achieved with a limit of 4.68 × 10−5 U mL−1. Furthermore, the method was adopted to screen the inhibitor of UDG and assay the activity of UDG in HeLa cell lysate. This method will offer a promising analysis tool for further biomedical research of UDG and clinical diagnosis.

Live Visualization of HIV-1 Proviral DNA Using a Dual-Color-Labeled CRISPR System.

HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mapped based on the colocalized signals of QDs in living cells. Compared to the existing zinc finger proteins and TALENs, the CRISPR system is much easier to operate and more efficient in imaging of internal genomic loci. Therefore, the proposed method could be not only a powerful tool for imaging proviral HIV-1, but also a versatile platform to image single genomic loci in living cells.

Chemoselective and Bioorthogonal Ligation Reactions. Concepts and Applications. 2 Volumes Edited by W. Russ Algar, Philip Dawson, and Igor L. Medintz.

Chemoselective and Bioorthogonal Ligation Reactions. Concepts and Applications. 2 Volumes Edited by W. Russ Algar, Philip Dawson, and Igor L. Medintz.

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide-peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA-templated ligation includes easy on-resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5-dimethoxy-2-nitrobenzaldehyde-based linker that allowed the photocleavable linkage of two bio-oligomers was developed.

Wavelength Dependence of Light-Induced Cycloadditions.

The wavelength-dependent conversion of two rapid photoinduced ligation reactions, i.e., the light activation of o-methylbenzaldehydes, leading to the formation of reactive o-quinodimethanes (photoenols), and the photolysis of 2,5-diphenyltetrazoles, affording highly reactive nitrile imines, is probed via a monochromatic wavelength scan at constant photon count. The transient species are trapped by cycloaddition with N-ethylmaleimide, and the reactions are traced by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The resulting action plots are assessed in the context of Beer-Lambert's law and provide combined with time-dependent density functional theory and multireference calculations an in-depth understanding of the underpinning mechanistic processes, including conical intersections. The π → π* transition of the carbonyl group of the o-methylbenzaldehyde correlates with a highly efficient conversion to the cycloadduct, showing no significant wavelength dependence, while conversion following the n → π* transition proceeds markedly less efficient at longer wavelengths. The influence of absorbance and reactivity has critical consequences for an effective reaction design: At high concentrations of o-methylbenzaldehydes (c = 8 mmol L-1), photoligations with N-ethylmaleimide (possible for λ ≤ 390 nm) are ideally performed at 330 nm, whereas at high light penetration regimes at lower concentrations (c = 0.3 mmol L-1), 315 nm irradiation leads to the highest conversion. Activation and trapping of 2,5-diphenyltetrazoles (possible for λ ≤ 322 nm) proceeds best at a wavelength shorter than 295 nm, irrespective of concentration.

Integrating Two Efficient and Specific Bioorthogonal Ligation Reactions with Natural Metabolic Incorporation in One Cell for Virus Dual Labeling.

Though techniques in bioorthogonal chemistry and metabolic incorporation have been developed over the past decade, it remains difficult to integrate different bioorthogonal reactions or metabolic incorporations into one system. In this report, the protein and DNA metabolic incorporations were combined with two bioorthogonal reactions in one cell to develop a facile and universal method for virus dual labeling. Azide and vinyl groups were introduced into the proteins or genomes of viruses, respectively, through the intrinsic biosynthesis of biomolecules, which were subsequently fluorescently labeled via copper-free click chemistry or alkene-tetrazine ligation reactions during natural propagation process in host cells. Both the envelope viruses and the capsid viruses could be labeled, and the dual labeling efficiency was more than 80%. The labeled progeny virions were structurally intact and fully infectious, and their fluorescence was strong enough to track single virions.

Current Status of Point-of-Care Testing for Human Immunodeficiency Virus Drug Resistance.

Healthcare delivery has advanced due to the implementation of point-of-care testing, which is often performed within minutes to hours in minimally equipped laboratories or at home. Technologic advances are leading to point-of-care kits that incorporate nucleic acid-based assays, including polymerase chain reaction, isothermal amplification, ligation, and hybridization reactions. As a limited number of single-nucleotide polymorphisms are associated with clinically significant human immunodeficiency virus (HIV) drug resistance, assays to detect these mutations have been developed. Early versions of these assays have been used in research. This review summarizes the principles underlying each assay and discusses strategic needs for their incorporation into the management of HIV infection.

Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures Using Orthogonal Ligations.

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4). LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation.

We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5'-flap endonuclease homologous to the 5'-exonuclease of DNA polymerase I. FenA incises a nicked 5' flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3' single strand is eliminated to create a 5'-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3'-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5' App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD+-dependent DNA ligases and especially by mycobacterial DNA ligases D and C.IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5'-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5' flaps, 5' nicks, and 5' App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease.

The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation

In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme-binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome c, CcmG, and the heme-ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants, we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that Cys-45 of CcmH and Cys-34 of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm and present a comprehensive model for thioreduction reactions that lead to heme-apocytochrome c ligation.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation.

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.

Maturation of Plastid c-type Cytochromes.

Cytochromes c are hemoproteins, with the prosthetic group covalently linked to the apoprotein, which function as electron carriers. A class of cytochromes c is defined by a CXXCH heme-binding motif where the cysteines form thioether bonds with the vinyl groups of heme. Plastids are known to contain up to three cytochromes c. The membrane-bound cytochrome f and soluble cytochrome c6 operate in photosynthesis while the activity of soluble cytochrome c6A remains unknown. Conversion of apo- to holocytochrome c occurs in the thylakoid lumen and requires the independent transport of apocytochrome and heme across the thylakoid membrane followed by the stereospecific attachment of ferroheme via thioether linkages. Attachment of heme to apoforms of plastid cytochromes c is dependent upon the products of the CCS (for cytochrome c synthesis) genes, first uncovered via genetic analysis of photosynthetic deficient mutants in the green alga Chlamydomonas reinhardtii. The CCS pathway also occurs in cyanobacteria and several bacteria. CcsA and CCS1, the signature components of the CCS pathway are polytopic membrane proteins proposed to operate in the delivery of heme from the stroma to the lumen, and also in the catalysis of the heme ligation reaction. CCDA, CCS4, and CCS5 are components of trans-thylakoid pathways that deliver reducing equivalents in order to maintain the heme-binding cysteines in a reduced form prior to thioether bond formation. While only four CCS components are needed in bacteria, at least eight components are required for plastid cytochrome c assembly, suggesting the biochemistry of thioether formation is more nuanced in the plastid system.

Simultaneous Identification of Ten Bacterial Pathogens Using the Multiplex Ligation Reaction Based on the Probe Melting Curve Analysis

Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.

Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.

The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.