Quantitative analysis of RNA by HPLC and evaluation of RT-dPCR for coronavirus RNA quantification.

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including open reading frame 1ab (ORF1ab), nucleocapsid protein (N) and envelope protein (E) gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme.

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

A Novel Alkaline Phosphatase/Phosphodiesterase, CamPhoD, from Marine Bacterium Cobetia amphilecti KMM 296.

A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S-1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S-1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.

Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities.

Mycobacterium smegmatis FenA is a nucleic acid phosphodiesterase with flap endonuclease and 5' exonuclease activities. The 1.8 Å crystal structure of FenA reported here highlights as its closest homologs bacterial FEN-family enzymes ExoIX, the Pol1 exonuclease domain and phage T5 Fen. Mycobacterial FenA assimilates three active site manganese ions (M1, M2, M3) that are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. We find via mutagenesis that the carboxylate contacts to all three manganese ions are essential for FenA's activities. Structures of nuclease-dead FenA mutants D125N, D148N and D208N reveal how they fail to bind one of the three active site Mn2+ ions, in a distinctive fashion for each Asn change. The structure of FenA D208N with a phosphate anion engaged by M1 and M2 in a state mimetic of a product complex suggests a mechanism for metal-catalyzed phosphodiester hydrolysis similar to that proposed for human Exo1. A distinctive feature of FenA is that it does not have the helical arch module found in many other FEN/FEN-like enzymes. Instead, this segment of FenA adopts a unique structure comprising a short 310 helix and surface ß-loop that coordinates a fourth manganese ion (M4).

Reversible Bioconjugation: Biodegradable Poly(phosphate)-Protein Conjugates.

Protein-polymer conjugates are widely used to improve the pharmacokinetic properties of therapeutic proteins. Commercially available conjugates employ poly(ethylene glycol) (PEG) as the protective polymer; however, PEG has a number of shortcomings, including non-biodegradability and immunogenicity, that call for the development of alternatives. Here, the synthesis of biodegradable poly(phosphate), that is, poly(ethyl ethylene phosphate) (PEEP), by organo-catalyzed anionic ring-opening polymerization exhibiting dispersity values Ð < 1.3 is reported. Polymers with molecular weights between 2000 and 33 200 g mol-1 are then ω-functionalized with a succinimidyl carbonate group and subsequently conjugated to model proteins. These are the first conjugates based on polyphosphates which degraded upon exposure to phosphodiesterase. As is the case for PEGylated therapeutics, residual in vitro activity of the PPEylated conjugates depends on the extent of protein modification. These results suggest that PEEP exhibits the desired properties of a biopolymer for use in next generation, fully degradable drug delivery systems.

Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides.

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5-30 Kb (∼1.6-10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.

Nudix hydrolases degrade protein-conjugated ADP-ribose.

ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR.

Synthesis and Biological Potential Assessment of 2-Substituted Quinazolin-4(3H)-ones as Inhibitors of Phosphodiesterase-I and Carbonic Anhydrase-II.

A library of twenty-five derivatives of 2-substituted quinazolin-4(3H)-ones 1-25 was synthesized and evaluated against phosphodiesterase-I (PDE) and carbonic anhydrase-II (CA). Compounds 17 (IC50 = 210.7 ± 2.62 µM), 16 (IC50 = 301.6 ± 1.18 µM), and 13 (IC50 = 458.13 ± 3.60 µM), selectively exhibited PDE inhibition while compounds 22 (IC50 = 61.33 ± 2.38 µM), 1 (IC50 = 108.30 ± 0.93 µM), and 21 (IC50 = 191.93 ± 2.72 µM), discriminatingly exhibited CA inhibition as compared to standards EDTA (IC50 = 277.69 ± 2.52 µM) and acetazolamide (IC50 = 0.12 ± 0.03 µM), for PDE and CA inhibitions, respectively. However, compound 15 was found to be active against both enzymes with the IC50 values 344.33 ± 4.32 µM and 20.94 ± 0.58 µM, for PDE and CA inhibitions, respectively. Remaining compounds were found to be inactive against both the enzymes. Structure-activity relationship studies are discussed herein.

C5-amino acid functionalized LNA: positively poised for antisense applications.

Incorporation of positively charged C5-amino acid functionalized LNA uridines into oligodeoxyribonucleotides (ONs) results in extraordinary RNA affinity, binding specificity and stability towards 3'-exonucleases.

Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction.

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.

Capillary monolithic bioreactor of immobilized snake venom phosphodiesterase for mass spectrometry based oligodeoxynucleotide sequencing.

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.

Fluorescence polarization biosensor based on an aptamer enzymatic cleavage protection strategy.

A novel fluorescence polarization (FP) aptasensing platform based on target-induced aptamer enzymatic cleavage protection is reported. The method relies on the FP analysis of the phosphodiesterase I mediated size variation of a dye-labeled aptamer. The tyrosinamide/antityrosinamide DNA aptamer couple was firstly tested as a model system to establish the proof-of-concept. In the absence of the target, the labeled aptamer was enzymatically cleaved into small DNA fragments, leading to a low FP signal. Upon tyrosinamide binding, the DNA substrate was partially protected against the enzymatic attack, leading to an increase in the fluorescence anisotropy response as a result of the higher average molecular volume of the weakly digested probe. The method was subsequently applied to two other systems, i.e., for the detection of ochratoxin A and adenosine. Such an approach was found to combine simplicity and general applicability features.

Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins.

Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 µm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.

Crystallization and preliminary X-ray crystallographic analysis of human autotaxin.

Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Šresolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, ß = 122.6°.

Location, location, location: a crystal-clear view of autotaxin saturating LPA receptors.

The interaction of autotaxin with its substrates leads to the production of lysophosphatidic acids (LPA), bioactive lipids with an emerging prominent role in inflammation and cancer. Two papers in this issue tell the previously unknown story of autotaxin, from substrate discrimination to highly efficient local delivery of LPA to target receptors.

A model for transition of 5'-nuclease domain of DNA polymerase I from inert to active modes.

Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented.

Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.

Autotaxin (ATX, also known as Enpp2) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine to generate lysophosphatidic acid (LPA), a lipid mediator that activates G protein-coupled receptors to evoke various cellular responses. Here, we report the crystal structures of mouse ATX alone and in complex with LPAs with different acyl-chain lengths and saturations. These structures reveal that the multidomain architecture helps to maintain the structural rigidity of the lipid-binding pocket, which accommodates the respective LPA molecules in distinct conformations. They indicate that a loop region in the catalytic domain is a major determinant for the substrate specificity of the Enpp family enzymes. Furthermore, along with biochemical and biological data, these structures suggest that the produced LPAs are delivered from the active site to cognate G protein-coupled receptors through a hydrophobic channel.

Large scale purification and characterization of recombinant human autotaxin/lysophospholipase D from mammalian cells.

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts.

BACKGROUND: Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis. CONCLUSION/SIGNIFICANCE: Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases.