From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor.

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Enhancement of antibody production against rabies virus by uridine 5'-triphosphate in mice.

Extracellular nucleotides such as adenosine 5'-triphospate (ATP) and uridine 5'-triphosphate (UTP) interact with P2 purinergic receptors on the surface of phagocytic cells and induce various physiological reactions. In this study, the production of antibody in mice immunized with an inactivated rabies vaccine containing these nucleotides was investigated. Injection of inactivated rabies vaccine with UTP, but not with ATP, induced significantly higher serum antibody production in mice. The enhancement of antibody production by UTP was inhibited by an anti-P2Y4 receptor antibody. In an air pouch experiment, UTP treatment increased the number of monocytes and macrophages infiltrating the pouch and up-regulated the gene expression of IL-4 and IL-13 in the regional lymph nodes. These results suggested that UTP admixed with rabies vaccine activates Th2 cells and induces a humoral immune response. Furthermore, the survival rate of mice immunized with a rabies vaccine admixed with UTP before rabies virus challenge was slightly higher than that of control mice. In conclusion, UTP can act as a vaccine adjuvant to enhance antibody production against the rabies virus in mice.

Functional implications from the Cid1 poly(U) polymerase crystal structure.

In eukaryotes, mRNA degradation begins with poly(A) tail removal, followed by decapping, and the mRNA body is degraded by exonucleases. In recent years, the major influence of 3'-end uridylation as a regulatory step within several RNA degradation pathways has generated significant attention toward the responsible enzymes, which are called poly(U) polymerases (PUPs). We determined the atomic structure of the Cid1 protein, the founding member of the PUP family, in its UTP-bound form, allowing unambiguous positioning of the UTP molecule. Our data also suggest that the RNA substrate accommodation and product translocation by the Cid1 protein rely on local and global movements of the enzyme. Supplemented by point mutations, the atomic model is used to propose a catalytic cycle. Our study underlines the Cid1 RNA binding properties, a feature with critical implications for miRNAs, histone mRNAs, and, more generally, cellular RNA degradation.

Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase.

TWINKLE is a nucleus-encoded human mitochondrial (mt)DNA helicase. Point mutations in TWINKLE are associated with heritable neuromuscular diseases characterized by deletions in the mtDNA. To understand the biochemical basis of these diseases, it is important to define the roles of TWINKLE in mtDNA metabolism by studying its enzymatic activities. To this end, we purified native TWINKLE from Escherichia coli. The recombinant TWINKLE assembles into hexamers and higher oligomers, and addition of MgUTP stabilizes hexamers over higher oligomers. Probing into the DNA unwinding activity, we discovered that the efficiency of unwinding is greatly enhanced in the presence of a heterologous single strand-binding protein or a single-stranded (ss) DNA that is complementary to the unwound strand. We show that TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs that interferes with unwinding in the absence of gp2.5 or ssDNA trap. Furthermore, only ssDNA and not double-stranded (ds)DNA competitively inhibits the annealing activity, although both DNAs bind with high affinities. This implies that dsDNA binds to a site that is distinct from the ssDNA-binding site that promotes annealing. Fluorescence anisotropy competition binding experiments suggest that TWINKLE has more than one ssDNA-binding sites, and we speculate that a surface-exposed ssDNA-specific site is involved in catalyzing DNA annealing. We propose that the strand annealing activity of TWINKLE may play a role in recombination-mediated replication initiation found in the mitochondria of mammalian brain and heart or in replication fork regression during repair of damaged DNA replication forks.

PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.

A stochastic two-dimensional model of intercellular Ca2+ wave spread in glia.

We describe a two-dimensional stochastic model of intercellular Ca(2+) wave (ICW) spread in glia that includes contributions of external stimuli, ionotropic and metabotropic P2 receptors, exo- and ecto-nucleotidases, second messengers, and gap junctions. In this model, an initial stimulus evokes ATP and UTP release from a single cell. Agonists diffuse and are degraded both in bulk solution and at cell surfaces. Ca(2+) elevation in individual cells is determined by bound agonist concentrations s and by number and features of P2 receptors summed with that generated by IP(3) diffusing through gap junction channels. Variability of ICWs is provided by randomly distributing a predetermined density of cells in a rectangular grid and by randomly selecting within intervals values characterizing the extracellular compartment, individual cells, and interconnections with neighboring cells. Variability intervals were obtained from experiments on astrocytoma cells transfected to express individual P2 receptors and/or the gap junction protein connexin43. The simulation program (available as Supplementary Material) permits individual alteration of ICW components, allowing comparison of simulations with data from cells expressing connexin43 and/or various P2 receptor subtypes. Such modeling is expected to be useful for testing phenomenological hypotheses and in understanding consequences of alteration of system components under experimental or pathological conditions.

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Kinetic studies of DNA cleavage reactions catalyzed by an ATP-dependent deoxyribonuclease on a 27-MHz quartz-crystal microbalance.

Catalytic DNA cleavage reactions by an ATP-dependent deoxyribonuclease (DNase) from Micrococcus luteus were monitored directly with a DNA-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the electrode at a nanogram level. Three steps in ATP-dependent DNA hydrolysis reactions, including (1) binding of DNase to the end of double-stranded DNA (dsDNA) on the QCM electrode (mass increase), (2) degradation of one strand of dsDNA in the 3' --> 5' direction depending on ATP (mass decrease), and (3) release of the enzyme from the nonhydrolyzed 5'-free-ssDNA (mass decrease), could be monitored stepwise from the time dependencies of QCM frequency changes. Kinetic parameters for each step were obtained as follows. The binding constant (K(a)) of DNase to the dsDNA was determined as (28 +/- 2) x 10(6) M(-)(1) (k(on) = (8.0 +/- 0.3) x 10(3) M (-)(1) s(-)(1) and k(off) = (0.29 +/-0.01) x 10(-)(3) s(-)(1)), and it decreased to (0.79 +/- 0.16) x 10(6) M(-)(1) (k'(on) = (2.3 +/- 0.2) x 10(3) M (-)(1) s(-)(1) and k'(off) = (2.9 +/- 0.1) x 10(-)(3) s(-)(1)) for the completely nonhydrolyzed 5'-free ssDNA. This is the reason the DNase bound to the dsDNA substrate can easily release from the nonhydrolyzed 5'-free-ssDNA after the complete hydrolysis of the 3' --> 5' direction of the complementary ssDNA. K(a) values depended on the DNA structures on the QCM, and the order of these values was as follows: the dsDNA having a 4-base-mismatched base-pair end (3) > the dsDNA having a 5' 15-base overhanging end (2) > the dsDNA having a blunt end (1) > the ssDNA having a 3'-free end (4) >> the ssDNA having a 5'-free end (5). Thus, DNase hardly recognized the free 5' end of ssDNA. Michaelis-Menten parameters (K(m) for ATP and k(cat)) of the hydrolysis process also could be obtained, and the order of k(cat)/K(m) was as follows: the dsDNA having a blunt end (1) approximately the dsDNA having a 4-base-mismatched base-pair end (3) > the ssDNA having a free 3' end (4) >> the ssDNA having a free 5' end (5). Thus, DNase could not recognize and not hydrolyze the free 5' end of ssDNA. The DNA hydrolysis reaction could be driven by dATP and GTP (purine base) as well as ATP, whereas the cleavage efficiency was very low driven with UTP, CTP (pyrimidine base), ADP, and AMP.

In vitro selection of human alpha-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP.

We synthesized 4'-thioUTP (1) and 4'-thioCTP (2) with the aim of developing new NTP analogs for in vitro selection. Since in vitro selection requires both in vitro transcription and reverse transcription, we examined the ability of 1 and 2 for in vitro selection by focusing on both steps. Incorporation of 1 and 2 by T7 RNA polymerase to give 4'-thioRNA proceeded well and was superior to those of the two sets of frequently used modified NTP analogs (2'-NH2dUTP and 2'-NH2dCTP, and 2'-FdUTP and 2'-FdCTP) for in vitro selection. In addition, reverse transcription of the resulting 4'-thioRNA into the complementary DNA in the presence of dNTPs also proceeded smoothly and precisely. With these successful results in hand, in vitro selection of human a-thrombin RNA aptamer using 1 and 2 is in progress.

Aminomodified nucleobases: functionalized nucleoside triphosphates applicable for SELEX.

5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP is reported in detail.

Hydrodynamic and spectroscopic studies of substrate binding to human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, plays a key role in the activation of therapeutic nucleoside analogues by their 5'-phosphorylation. The structure of human dCK is still not known and the current work was undertaken to determine its oligomeric and secondary structure. Biophysical studies were conducted with purified recombinant human dCK. The Mr determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 +/- 1,000, indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with ellipticity values of -11,900 +/- 300 and -12,500 +/- 300 deg x cm2 x dmol(-1), respectively, indicating the presence of approximately 40% alpha-helix and 50% beta-structure. Circular Dichroism studies in the aromatic and far-ultraviolet range and UV difference spectroscopy indicated that binding of substrates to dCK reduced its alpha-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence demonstrated that deoxycytidine (the phosphate acceptor) and ATP (the phosphate donor) bound to different sites on dCK and fluorescence quenching revealed bimodal binding of deoxycytidine and unimodal binding of ATP. Spectroscopic studies indicated that substrate binding induced conformational changes, with the result that dCK exhibited different affinities for various substrates. These results are consistent with a random bi-bi kinetic mechanism of phosphorylation of dCyd with either ATP or UTP.

Fluorescent labelling of cRNA for microarray applications.

Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP (aa-UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy-UTPs. The presence of dimethylsulfoxide during coupling of aa-modified cRNA with N-hydroxysuccinimide-modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2- to 3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.

Methods to enhance signal using isotopic in situ hybridization.

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

Hybrid selection with cDNA-silica.

A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 micrograms/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29-162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.