Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer

In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer. The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments. After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5′-end only. Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used. As an example we report the amplification of the era gene from Lactococcus lactis.     

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental ¹³C/¹⁵N/²H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively ¹³C₉/¹⁵N₃/²H_{(1′,2″,3′,4′,5′,5″)}-dC and ¹³C₉/¹⁵N₂/²H_{(1′,2″,3′,4′,5′,5″)}-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective ²H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1′–H2′ and C2′–H2′ resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.     

Tapping diversity lost in transformations—in vitro amplification of ligation reactions

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>10⁹ transformants in Escherichia coli and 10⁵-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.     

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3′-untranslated region (3′-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3′-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3′-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3′-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1–100 of 3′-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H₂O₂ led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H₂O₂-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H₂O₂ on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H₂O₂ on AT1R mRNA.     

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V_κ and V_H genes with newly designed primer sets. Initially, we amplified V_κ and V_H genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V_κ and V_H genes from the framework region 1 to the joining region. The V_κ and V_H genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.     

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

We set out to gain deeper insight into the potential of antibody light chain variable domains (V_Ls) as immunotherapeutics. To this end, we generated a naïve human V_L phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (V_Hs), we isolated a diversity of V_L domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating V_L repertoires are a rich source of non-aggregating domains. In addition, the V_Ls demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human V_Hs revealed that the V_Ls had similar overall profiles with respect to melting temperature (T_m), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of V_Ls did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T_ms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of V_Ls. Human V_Ls and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered V_Ls may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.     

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V_HH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V_HH from a humanized-camel VH/V_HH display library. VH/V_HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V_HH hallmark and were designated V_HH6 and V_HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V_HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V_HH6 and V_HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V_HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.     

A novel single-stranded DNA-specific 3′–5′ exonuclease,Thermus thermophilus exonuclease I,is involved in several DNA repair pathways

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5′–3′ and 3′–5′ ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3′–5′ ssExo, resulted in significant increase in the sensitivity to H₂O₂ and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5′–3′ ssExo, showed little effect on the sensitivity to H₂O₂, but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3′–5′ ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3′–5′ ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.     

Synthetic antibodies designed on natural sequence landscapes

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V_H) and two kappa (V_κ) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V_H and V_κ sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6 × 10¹⁰ transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0 × 10⁵ clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.     

Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H₂ uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (ΔG′) and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (ΔG′ = −17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (ΔG′ = −3.8 kJ/mol, v = 0.12 mM/h). The ΔG′ value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable ΔG′ resulted from the close coupling of the rates of butyrate and H₂ oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H₂ sink. When the specific activity of H₂ consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H₂ pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H₂ consumption, not the type of H₂ consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V_max.     

Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC₅₀ values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3′–5′ exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5′–3′ exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis.     

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus

An NAD⁺-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg²⁺ or Mn²⁺ as the metal cofactor. Ca²⁺ and Ni²⁺ mainly support formation of DNA–adenylate intermediates. The catalytic cycle is characterized by a low k_cat value of 2 min^–1 with concomitant accumulation of the DNA–adenylate intermediate when Mg²⁺ is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A ≤ G/C < C/G < A/T on both the 3′- and 5′-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3′-side of the nick junction. The requirement of 3′ complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3′- or 5′-side of the nick. Furthermore, most of the unligatable 3′ mismatched base pairs prohibit formation of the DNA–adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5′-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.     

Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6–8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3′-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3′-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 10⁸ clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in E. coli, enabling large number of different clones to be rapidly generated.     

Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning

Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ~10-fold when the genomic DNA is sheared to 100–200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector’s targeting hooks when they are located at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ~20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages

The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (V_H) chain and light chain (V_L), then coupled with highly conserved regions of the heavy chain domain 1 (C_H1) and the light chain (C_L), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis.     

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase–adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8–9 nt on the 3′-hydroxyl (3′-OH) side of the nick and 11–12 nt on the 5′-phosphate (5′-PO₄) side. Here we establish the minimal length requirements for ligatable 3′-OH and 5′-PO₄ strands at the nick (6 nt) and describe a new crystal structure of the ligase–adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase–adenylate bound to sulfate (a mimetic of the nick 5′-PO₄) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small ‘pluripotent’ ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein–protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.