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Metal-mediated base pairs enable a site-specific incorporation of transition metal ions into nucleic acid structures. The resulting nucleic acid-metal complex conjugates are of interest in the context of functionalized nucleic acids, as they bear metal-based functionality. It is desirable to devise nucleic acids with an externally triggered metal-binding affinity, as this may allow regulating this functionality. Toward this end, a caged deoxyribonucleoside analog HNPP was devised for the site-specific binding of copper(II) ions upon irradiation by light, based on the ligand 3-hydroxy-2-methylpyridin-4(1H)-one (H) and the photocleavable 2-(2-nitrophenyl)propoxy protecting group (NPP). The formation of both H-Cu(II)-H homo base pairs and H-Cu(II)-X hetero base pairs (involving a second artificial deoxyribonucleoside X, based on imidazole-4-carboxylate) was achieved upon irradiation of DNA duplexes bearing the respective HNPP:HNPP or HNPP:X mispairs in the presence of copper(II) ions. The H-Cu(II)-X pair shows an exceptional DNA duplex stabilization of up to 43 °C upon its formation, exceeding that of the H-Cu(II)-H pair. It therefore represents one of the most stabilizing Cu(II)-mediated base pairs reported so far. Our findings expand the scope of light-triggered metal-mediated base pair formation by introducing a copper(II)-binding ligand.

Human concentrative nucleoside transporters (CNTs) are responsible for cellular uptake of ribonucleosides; however, although it is important to better characterize CNT-subtype specificity to understand the systemic disposition of deoxyribonucleosides (dNs) and their analogs, the involvement of CNTs in transporting dNs is not fully understood. In this study, using COS-7 cells that transiently expressed CNT1, CNT2, or CNT3, we investigated if CNTs could transport not only ribonucleosides but also dNs, i.e., 2ʹ-deoxyadenosine (dAdo), 2ʹ-deoxyguanosine (dGuo), and 2ʹ-deoxycytidine (dCyd). The cellular uptake study demonstrated that dAdo and dGuo were taken up by CNT2 but not by CNT1. Although dCyd was taken up by CNT1, no significant uptake was detected in COS-7 cells expressing CNT2. Similarly, these dNs were transported by CNT3. The apparent Km values of their uptake were as follows: CNT1, Km ＝ 141 μM for dCyd; CNT2, Km ＝ 62.4 μM and 54.9 μM for dAdo and dGuo, respectively; CNT3, Km ＝ 14.7 μM and 34.4 μM for dGuo and dCyd, respectively. These results demonstrate that CNTs contribute not only to ribonucleoside transport but also to the transport of dNs. Moreover, our data indicated that CNT1 and CNT2 selectively transported pyrimidine and purine dNs, respectively, and CNT3 was shown to transport both pyrimidine and purine dNs.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with 1,3‐diaza‐2‐oxophenoxazine (BNAP (BH)) was previously reported. Herein, a novel BH analogue, 2′,4′‐BNA/LNA with 9‐(2‐aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine (G‐clamp), named BNAP‐AEO (BAEO), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104‐, 152‐, and 11‐fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′‐deoxycytidine (C), 2′,4′‐BNA/LNA with 5‐methylcytosine (L), or 2′‐deoxyribonucleoside with G‐clamp (PAEO), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3′‐exonuclease than ODNs with phosphorothioate linkages., Stability by design: Artificial nucleic acids are widely used and in some applications require excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with G‐clamp (BNAP‐AEO) is synthesized and incorporated into oligodeoxynucleotides (ODNs). These modified ODNs show high thermal stabilities toward complementary DNA and RNA.

Stabilization of DNA is beneficial for many applications in the fields of DNA therapeutics, diagnostics, and materials science. Now, this phenomenon is studied on heterochiral DNA, an autonomous DNA recognition system with complementary strands in α‐D and β‐D configuration showing parallel strand orientation. The 12‐mer heterochiral duplexes were constructed from anomeric (α/β‐D) oligonucleotide single‐strands. Purine‐2,6‐diamine and 8‐aza‐7‐deaza‐7‐bromopurine‐2,6‐diamine 2′‐deoxyribonucleosides having the capability to form tridentate base pairs with dT were used to strengthen the stability of the dA–dT base pair. T m data and thermodynamic values obtained from UV melting profiles indicated that the 8‐aza‐7‐deaza 2′‐deoxyribonucleoside decorated with a bromo substituent is so far the most efficient stabilizer for heterochiral DNA. Compared with that, the stabilizing effect of the purine‐2,6‐diamine 2′‐deoxyribonucleoside is low. Global changes of helix structures were identified by circular dichroism (CD) spectra during melting., Base pair stabilization: Heterochiral parallel duplexes represent an autonomous recognition system constructed from anomeric (α/β‐D) oligonucleotides. The naturally occurring purine‐2,6‐diamine and the artificial 8‐aza‐7‐deaza‐7‐bromopurine nucleosides 1 and 2 strengthen the dA–dT base pair. The 8‐aza‐7‐deazapurine nucleoside decorated with a bromo substituent is so far the most efficient stabilizer for this pairing system.

Unlike latent cells, cancer cells supply deoxyribonucleoside triphosphates to cells continuously and thereby, prop up the uncontrolled cancer growth. Pyrimidine has been concerned in the separation of leukemic cells, known as adenine bioisosteres, as well as its biological activities, especially its anticancer properties. In this context, a novel series of N5-(3-substituted benzylidene)-N4-phenyl pyrimidine-4,5-diamine [5A-5F] / N5-(2-substituted benzylidene)-N2,N2-dimethyl-N4-phenyl pyrimidine-2,4,5-triamine [5a-5f] were synthesized by using the starting ingredient formimidamide/4-(dimethylamino) benzimidamide and sodium ethoxide. The synthesized compounds were characterized by IR, 1H NMR, and Mass spectral analyses and screened for their biological studies. In the present study, pyrimidine derivatives and their insilico modeling were done by using c-Src kinase and p38 MAP kinase complex followed by the evaluation of their anticancer activity. The screening of synthesized scaffolds possessed significant activity against HeLa cell lines and showed similar activity compared to standard Cisplatin. Among all the synthesized compounds, N5-(4-hydroxybenzylidene)-N4-phenyl pyrimidine-4,5-diamine 5A, N5-benzylidene-N4-phenylpyrimidine-4,5-diamine 5C, N5-benzylidene-N2, N2-dimethyl-N4-phenyl pyrimidine-2,4,5-triamine 5c, and N5-(4-methoxy benzylidene)-N4-phenyl pyrimidine-4,5-diamine 5E showed the highest significant anticancer activity. Based on the study, it was observed that substituents such as 4-hydroxy, 4-fluoro, 4-nitro, 4-methoxy, and 4-methyl are important to produce a biological effect. Moreover, among these substituents, N5-(4-hydroxybenzylidene)-N4-phenyl pyrimidine-4,5-diamine exhibited the highest anticancer activity followed by unsubstituted derivative N5-benzylidene-N4-phenylpyrimidine-4,5-diamine, which delivered significant anticancer activity.

Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, catalyses reversible transfer of the γ phosphate from nucleoside triphosphates to nucleoside diphosphates and functions to maintain the pools of ribonucleotides and deoxyribonucleotides in the cell. As even a minor imbalance in the nucleotide pools can be mutagenic, NDK plays an antimutator role in maintaining genome integrity. However, the mechanism of the antimutator roles of NDK is not completely understood. In addition, NDKs play important roles in the host-pathogen interactions, metastasis, gene regulation, and various cellular metabolic processes. To add to these diverse roles of NDK in cells, a recent study now reveals that NDK may even confer mutator phenotypes to the cell by acting on the damaged deoxyribonucleoside diphosphates that may be formed during the oxidative stress. In this review, we discuss the roles of NDK in homeostasis of the nucleotide pools and genome integrity, and its possible implications in conferring growth/survival fitness to the organisms in the changing environmental niches.

3′-N-(2-Thio-1,3,2-oxathiaphospholane) derivatives of 5′-O-DMT-3′-amino-2′,3′-dideoxy-ribonucleosides (NOTP-N), that bear a 4,4-unsubstituted, 4,4-dimethyl, or 4,4-pentamethylene substituted oxathiaphospholane ring, were synthesized. Within these three series, NOTP-N differed by canonical nucleobases (i.e., AdeBz, CytBz, GuaiBu, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare NNPSN′ dinucleotides (3), as well as short P-stereodefined oligo(deoxyribonucleoside N3′→O5′ phosphoramidothioate)s (NPS-) and chimeric NPS/PO- and NPS/PS-oligomers. The condensation reaction for NOTP-N monomers was found to be 5–6 times slower than the analogous OTP derivatives. When the 5′-end nucleoside of a growing oligomer adopts a C3′-endo conformation, a conformational ‘clash’ with the incoming NOTP-N monomer takes place, which is a main factor decreasing the repetitive yield of chain elongation. Although both isomers of NNPSN′ were digested by the HINT1 phosphoramidase enzyme, the isomers hydrolyzed at a faster rate were tentatively assigned the RP absolute configuration. This assignment is supported by X-ray analysis of the protected dinucleotide DMTdGiBuNPSMeTOAc, which is P-stereoequivalent to the hydrolyzed faster P-diastereomer of dGNPST.

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs' analogs have synthesized by structural changes of nucleoside's susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl - analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π-π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.

All four iodinated 2'-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.

The polyanionic phosphodiester backbone of nucleic acids contributes to high nuclease sensitivity and low cellular uptake and is therefore a major obstacle to the biological application of native oligonucleotides. Backbone modifications, particularly charge alterations is a proven strategy to provide artificial oligonucleotides with improved properties. Here, we describe the synthesis of a new type of oligonucleotide analogues consisting of a morpholino and a ribo- or deoxyribonucleoside in which the 5′-amino group of the nucleoside unit provides the nitrogen of the morpholine ring. The synthetic protocol is compatible with trityl and dimethoxytrityl protecting groups and azido functionality, and was extended to the synthesis of higher oligomers. The chimeras are positively charged in aqueous medium, due to the N-alkylated tertiary amine structure of the morpholino unit.

Background The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas’ disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside ‘hit’ led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3′-deoxy-β-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. Objectives To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. Results Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. Conclusions Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

Hydroxycamptothecin (SN38) is a natural plant extract isolated from Camptotheca acuminate. It has a broad spectrum of anticancer activity through inhibition of DNA topoisomerase I, which could affect DNA synthesis and lead to DNA damage. Thus, the action of SN38 against cancers could inevitably affect endogenous levels of ribonucleotide (RNs) and deoxyribonucleotide (dRNs) that play critical roles in many biological processes, especially in DNA synthesis and repair. However, the exact impact of SN38 on RNs and dRNs is yet to be fully elucidated. In this study, we evaluated the anticancer effect and associated mechanism of SN38 in human colorectal carcinoma HCT 116 cells. As a result, SN38 could decrease the cell viability and induce DNA damage in a concentration-dependent manner. Furthermore, cell cycle arrest and intracellular nucleotide metabolism were perturbed due to DNA damage response, of which ATP, UTP, dATP, and TTP may be the critical metabolites during the whole process. Combined with the expression of deoxyribonucleoside triphosphates synthesis enzymes, our results demonstrated that the alteration and imbalance of deoxyribonucleoside triphosphates caused by SN38 was mainly due to the de novo nucleotide synthesis at 24 h, and subsequently the salvage pathways at 48 h. The unique features of SN38 suggested that it might be recommended as an effective supplementary drug with an anticancer effect.

WSL8 encoding a deoxyribonucleoside kinase (dNK) that catalyzes the first step in the salvage pathway of nucleotide synthesis plays an important role in early chloroplast development in rice. The chloroplast is an organelle that converts light energy into chemical energy; therefore, the normal differentiation and development of chloroplast are pivotal for plant survival. Deoxyribonucleoside kinases (dNKs) play an important role in the salvage pathway of nucleotides. However, the relationship between dNKs and chloroplast development remains elusive. Here, we identified a white stripe leaf 8 (wsl8) mutant that exhibited a white stripe leaf phenotype at seedling stage (before the four-leaf stage). The mutant showed a significantly lower chlorophyll content and defective chloroplast morphology, whereas higher reactive oxygen species than the wild type. As the leaf developed, the chlorotic mutant plants gradually turned green, accompanied by the restoration in chlorophyll accumulation and chloroplast ultrastructure. Map-based cloning revealed that WSL8 encodes a dNK on chromosome 5. Compared with the wild type, a C-to-G single base substitution occurred in the wsl8 mutant, which caused a missense mutation (Leu 349 Val) and significantly reduced dNK enzyme activity. A subcellular localization experiment showed the WSL8 protein was targeted in the chloroplast and its transcripts were expressed in various tissues, with more abundance in young leaves and nodes. Ribosome and RNA-sequencing analysis indicated that some components and genes related to ribosome biosynthesis were down-regulated in the mutant. An exogenous feeding experiment suggested that the WSL8 performed the enzymic activity of thymidine kinase, especially functioning in the salvage synthesis of thymidine monophosphate. Our results highlight that the salvage pathway mediated by the dNK is essential for early chloroplast development in rice.

Background: Sufficient supply of deoxyribonucleoside triphosphates (dNTPs) is required for the uncontrolled replication of cancers. The current study aimed to investigate the biological and clinical role of ribonucleotide reductase subunit M2 (RRM2), a key enzyme regulating the dNTP pool, in clear-cell renal cell carcinoma (ccRCC). Methods: The expression of RRM2 on disease progression and patient outcome was assessed in ccRCC. Then, the effect of RRM2 inhibition on renal cell carcinoma (RCC) growth using siRNA or Triapine, an RRM2-specific inhibitor, was characterized in RCC cell lines. Results: The expression of RRM2 was up-regulated in ccRCC tissues as compared to the normal tissues. Patients with high RRM2 expression tend to have advanced pT stages, high Fuhrman grades, and shortened overall survival (OS). RRM2-siRNAs or Triapine significantly inhibited the cell growth by inducing G0/G1 cell cycle arrest in RCC cells through the attenuation of dNTP pool. Conclusions: The current results provided evidence that RRM2 might act as a novel target for ccRCC, and exploration of nonnucleoside, reversible, small-molecule inhibitors against RRM2 could be promising.

Alkaline phosphatase (ALP) is a vital hydrolysis enzyme in phosphate metabolism, which catalyzes the hydrolysis of phosphate ester groups in proteins, nucleic acids, and other small molecules. Meanwhile, abnormal ALP expression is associated with occurrence and development of many diseases. Terminal deoxynucleotidyl transferase (TdT) is a widely used tool enzyme in many fields, which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3′-OH termini of ssDNA in a template-free manner. In this work, we designed a versatile, convenient, label-free, and highly sensitive fluorescence enhancing assay for ALP activity detection based on the characteristics of ALP, TdT, and thymine-HgII-thymine (T-Hg2+-T) structure. In the presence of ALP, the 3′-phosphoryl end of the ssDNA-p was hydrolyzed to hydroxyl group, followed by addition of a poly-T tail on its 3′ terminal hydroxyl in the mixing solution containing both TdT and dTTPs. Then, the DNA with poly-T tail could interact with Hg2+ to form the stable T-Hg2+-T mediated metallo DNA duplex, which enhanced the fluorescence intensity of the SG. Under optimal conditions, the proposed system was employed for quantitatively monitoring ALP activity with a dynamic range of 0–2500 mU mL−1, and the actual detection limit could be down to 0.025 mU mL−1. And the determination of ALP activity in human serum samples and MCF-7 cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis. Meanwhile, this system could also be applied to both TdT and Hg2+ detection.

The use of imidazole-4-carboxylate (X) as an artificial nucleobase in metal-mediated base pairing is reported. Towards this end, the corresponding deoxyribonucleoside was synthesized and structurally characterized as its sodium salt (sodium 1,2-dideoxy-1-(4-carboxyimidazol-1-yl)- d -ribofuranose). The deoxyribonucleoside was incorporated into different DNA duplexes (parallel-stranded and antiparallel-stranded), and their Cu(II)- and Ag(I)-binding behavior was investigated. It was shown that both X–Cu(II)–X and X–Ag(I)–X base pairs can be formed, with the former being more stabilizing than the latter. The formation of an X–Cu(II)–X base pair is accompanied by an increase in the duplex melting temperature of approximately 20 °C for antiparallel-stranded duplexes and of 12 °C for the parallel-stranded duplex under investigation. Imidazole-4-carboxylate represents the first imidazole-based nucleoside for Cu(II)-mediated base pairing. Moreover, it is the smallest nucleoside known to form stable Cu(II)-mediated base pairs. Structures of the X–Cu(II)–X and X–Ag(I)–X base pairs are proposed, too, based on molecular structures obtained using the model nucleobase 1-benzyl-1H-imidazole-4-carboxylate.

Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3′-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.

Loss‐of‐function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK‐deficient, patient‐derived fibroblasts and myoblasts. CERC‐913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC‐913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK‐deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC‐913 increased mtDNA content in DGUOK‐deficient hepatocytes up to 2.4‐fold after 4 days of treatment in a dose‐dependent fashion, which was significantly more effective than dGMP at similar concentrations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC‐913 treatment can improve mtDNA content in this model.

Background/aim The expression of corticotropin-releasing hormone (CRH)-related peptides involved in stress response in colorectal cancer has been reported. We examined the involvement of CRH-related peptides in cellular stress caused by anticancer drugs in colorectal cancer. Materials and methods Changes in the expression levels of CRH-related peptides and their receptors in HCT116, DLD-1, and SW480 cell lines after fluorouracil (5-FU) loading were evaluated. Effects of the receptor antagonist against DNA synthesis disorder caused by 5-FU and SN-38 were evaluated using the 3H-labeled deoxyribonucleoside incorporation assay. Results No changes in the mRNA expression levels of CRH-related peptides (UCN and UCN2) -and their receptors (CRHR1 and CRHR2) were observed. Addition of antagonists to cells with DNA synthesis disorder caused by 5-FU and SN-38 showed no differences in the incorporation of 3H-labeled deoxyribonucleoside. Conclusion CRH-related peptides showed no effect on the stress response to anticancer drugs nor on DNA synthesis disorder in colorectal cancer.

Brain-infiltrating leukocytes contribute to multiple sclerosis (MS) and autoimmune encephalomyelitis and likely play a role in traumatic brain injury, seizure, and stroke. Brain-infiltrating leukocytes are also primary targets for MS disease-modifying therapies. However, no method exists for noninvasively visualizing these cells in a living organism. 1-(2′-deoxy-2′-(18)F-fluoroarabinofuranosyl) cytosine ((18)F-FAC) is a PET radiotracer that measures deoxyribonucleoside salvage and accumulates preferentially in immune cells. We hypothesized that (18)F-FAC PET could noninvasively image brain-infiltrating leukocytes. Methods: Healthy mice were imaged with (18)F-FAC PET to quantify if this radiotracer crosses the blood–brain barrier (BBB). Experimental autoimmune encephalomyelitis (EAE) is a mouse disease model with brain-infiltrating leukocytes. To determine whether (18)F-FAC accumulates in brain-infiltrating leukocytes, EAE mice were analyzed with (18)F-FAC PET, digital autoradiography, and immunohistochemistry, and deoxyribonucleoside salvage activity in brain-infiltrating leukocytes was analyzed ex vivo. Fingolimod-treated EAE mice were imaged with (18)F-FAC PET to assess if this approach can monitor the effect of an immunomodulatory drug on brain-infiltrating leukocytes. PET scans of individuals injected with 2-chloro-2′-deoxy-2′-(18)F-fluoro-9-β-d-arabinofuranosyl-adenine ((18)F-CFA), a PET radiotracer that measures deoxyribonucleoside salvage in humans, were analyzed to evaluate whether (18)F-CFA crosses the human BBB. Results: (18)F-FAC accumulates in the healthy mouse brain at levels similar to (18)F-FAC in the blood (2.54 ± 0.2 and 3.04 ± 0.3 percentage injected dose per gram, respectively) indicating that (18)F-FAC crosses the BBB. EAE mice accumulate (18)F-FAC in the brain at 180% of the levels of control mice. Brain (18)F-FAC accumulation localizes to periventricular regions with significant leukocyte infiltration, and deoxyribonucleoside salvage activity is present at similar levels in brain-infiltrating T and innate immune cells. These data suggest that (18)F-FAC accumulates in brain-infiltrating leukocytes in this model. Fingolimod-treated EAE mice accumulate (18)F-FAC in the brain at 37% lower levels than control-treated EAE mice, demonstrating that (18)F-FAC PET can monitor therapeutic interventions in this mouse model. (18)F-CFA accumulates in the human brain at 15% of blood levels (0.08 ± 0.01 and 0.54 ± 0.07 SUV, respectively), indicating that (18)F-CFA does not cross the BBB in humans. Conclusion: (18)F-FAC PET can visualize brain-infiltrating leukocytes in a mouse MS model and can monitor the response of these cells to an immunomodulatory drug. Translating this strategy into humans will require exploring additional radiotracers.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Summary The anti-leukaemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphates (dNTPs), protected cells against the effects of dNTP imbalances. SAMHD1-deficient cells induced intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine acted synergistically to kill cells lacking SAMHD1. Using mass cytometry, we found that these compounds killed SAMHD1-deficient malignant cells from patients with chronic lymphocytic leukaemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation were unaffected. We therefore propose to use forodesine as a precision medicine for leukaemia, stratifying patients by SAMHD1 genotype or expression. Highlights SAMHD1-deficient cells die upon exposure to deoxyribonucleosides (dNs) Deoxyguanosine (dG) is the most toxic dN, inducing apoptosis in cells lacking SAMHD1 SAMHD1-mutated leukaemic cells can be killed by dG and the PNP-inhibitor forodesine In Brief SAMHD1 degrades deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Davenne et al. found that SAMHD1 protects cells against dNTP imbalances. Exposure of SAMHD1-deficient cells to deoxyguanosine (dG) results in increased intracellular dGTP levels and subsequent apoptosis. This can be exploited to selectively kill cancer cells that acquired SAMHD1 mutations.

4-Cyanoindole-2'-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2'-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase.

The synthesis and characterization of a universal and fluorescent nucleoside, 4-cyanoindole-2ʹ-deoxyribonucleoside (4CIN), and its incorporation into DNA is described. 4CIN is a highly efficient fluorophore with quantum yields >0.90 in water. When incorporated into duplex DNA, 4CIN pairs equivalently with native nucleobases and has uniquely high quantum yields ranging from 0.15–0.31 depending on sequence and hybridization contexts, and surpassing that of 2-aminopurine, the prototypical nucleoside fluorophore. 4CIN constitutes a new isomorphic nucleoside for diverse applications.

Background Nucleoside triphosphates participate in fundamental cellular processes as building blocks of DNA and RNA, energy carriers, and cofactors in enzymatic reactions, and their balance is tightly regulated. Here, we established a simultaneous and absolute quantification method for eight nucleoside triphosphates using liquid chromatography–triple quadrupole tandem mass spectrometry and hydrophilic interaction chromatography. Our method was successfully applied to the extract of human acute myeloid leukemia Molm-13 cells. Results Levels of ribonucleoside triphosphates (2.07 × 108–2.29 × 109 molecules/cell) in Molm-13 cells were two orders of magnitude higher than those of deoxyribonucleoside triphosphates (1.72 × 106–1.40 × 107 molecules/cell). Exposure of Molm-13 cells for 24 h to thymidine, a nucleotide imbalance inducer, increased the levels of cellular dTTP, dGTP, and dATP and decreased only dCTP, resulting in significant inhibition of cell proliferation. Conclusion Our quantification method for nucleoside triphosphates revealed the quantitative relationship between the arrest of cell proliferation and the imbalance of nucleoside triphosphates in thymidine-treated Molm-13 cells. Owing to the short run time (15 min/run), broad adaptability, and throughput performance, we believe that our method is a powerful tool for not only genetic and molecular biology research but also for studying the mechanism of genotoxic compounds and anti-cancer or anti-virus drugs, drug screening, clinical studies, and other fields.

(3,4-Dihydroxybut-1-ynyl)uracil, -cytosine and -7-deazaadenine 2'-deoxyribonucleoside triphosphates (dNTPs) were prepared by direct aqueous Sonogashira cross-coupling of halogenated dNTPs with dihydroxybut-1-yne and converted to 3,4-dihydroxybutyl dNTPs through catalytic hydrogenation. Sodium periodate oxidative cleavage of dihydroxybutyl-dUTP gave the desired aliphatic aldehyde-linked dUTP, whereas the oxidative cleavage of the corresponding deazaadenine dNTP gave a cyclic aminal. All dihydroxyalkyl or -alkynyl dNTPs and the formylethyl-dUTP were good substrates for DNA polymerases and were used for synthesis of diol- or aldehyde-linked DNA. The aldehyde linked DNA was used for the labelling or bioconjugations through hydrazone formation or reductive aminations.

An ultrasensitive electrochemical biosensor was developed for detection of T4 polynucleotide kinase (T4 PNK) activity based on titanium dioxide nanotubes (TiO2 NTs) and a rolling circle amplification (RCA) strategy. In this study, the immobilized T4 PNK substrate probe with a 5′ terminus hydroxyl was phosphorylated by T4 PNK in the presence of adenosine triphosphate (ATP), and the resulting 5-phosphoryl can be linked with the TiO2 NTs and further conjugated with the phosphate-labeled primer. RCA was initiated by adding circular template, phi29 DNA polymerase and deoxyribonucleoside 5-triphosphate mixture (dNTPs). Biotin-labeled probes are chosen as a signal indicator by strong biotin–streptavidin interaction and the high loading of horseradish peroxidase–streptavidin (HRP–SA) for electrochemical signal generation and amplification. A dual-signaling amplification strategy has been established, which exhibited an excellent performance with a wide linear range from 0.0001–15 U mL−1 and a low detection limit of 0.00003 U mL−1 for T4 PNK detection. The inhibition effect of (NH4)2SO4 on the activity of T4 PNK is also evaluated. This new dual-signaling electrochemical biosensor can be used for the detection of the activity and inhibition of other nucleic acid enzymes.

The endogenous epigenetic marker 5-formylcytosine (5 fC) is introduced by 5-methylcytosine (5 mC) oxidation under action of enzyme oxidation, and plays an important role in many life activities. Since the content of 5 fC in mammalian tissues and cells is very low, it is necessary to exploit a sensitive and specific detection method to further understand the function of 5 fC. In this work, a sensitively and selectively photoelectrochemical (PEC) biosensor was developed for 5-formyl-2′-deoxycytidine (5fdC) detection. CuBi2O4/ZnPc@WS2 was used as photoactive material, where the formed ternary heterojunction structure greatly enhanced the PEC response and increased the detection sensitivity. Positively charged polyethyleneimine (PEI) was employed as 5fdC recognition and capture unit, where the amine group on PEI specifically reacted with aldehyde group of 5fdC to form stable amide bond. 4-Carboxyphenylboronic acid (4-CPBA) was adopted as crosslinker for 5fdC and amino functionalized CuBi2O4 based on the covalent interaction between 1,3-diol bond on 5fdC and boric acid structure on 4-CPBA, and the covalent interaction between –COOH on 4-CPBA and –NH2 on amino functionalized CuBi2O4. On the basis of the positive synergistic effect of ZnPc and CuBi2O4 on improving the photoelectric performance of WS2, the separation of photo-generated electron-hole pairs in semiconductors were promoted, and the examination range was expanded from 0.1 to 500 nM, and the detection limit was 0.0483 nM (3σ). Based on the unique covalent reaction between –NH2 and –CHO, the PEC biosensor has excellent detection sensitivity, and can even separate 5fdC from 5-methylcytosine deoxyribonucleoside and 5-hydroxymethylcytosine deoxyribonucleoside. The effect of antibiotics and heavy metals on the 5fdC content in wheat tissue genome has also been further investigated using this sensor.

Cell apoptosis detection is vital for biological analysis and clinical application; some detection assays are already commercially available. However, it is still far from perfect and needs further improvement for less cost, time-consuming and operation demanding. TUNEL, a high market share cell apoptosis assay, depends on adulteration fluorescent labelling dUTP by terminal deoxynucleotidyl transferase(TdT) which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3'-OH terminal of ssDNA with a template-free manner. Based on our previous work, we adopted a label-free strategy to reduce the cost and operation maintenance of TUNEL and developed a facile, rapid, convenient and in-situ assay for cell apoptosis.

Analyses of nucleic acid amplification products are generally performed by the complicated gel electrophoresis or costly florescence method. In this work, colorimetric detection of phosphate ions (Pis) generated in the amplification reaction was proposed for user-friendly and low-cost detection of nucleic acid products. To overcome the interference from nonspecific Pis, the effects of deoxyribonucleoside triphosphates (dNTPs) and Mg2+ concentrations, thermal cycle and initial template were investigated. The results showed that the yields of nonspecific Pis were constant, reproducible, and was the same at the presence/absence of target template, suggesting the potential to simply measure and subtract the interference. A colorimetric reader was developed to detect Pis in the amplification tube. It revealed that the unique structure of the tube would diverge, merge and reflect the transmitting light depending on the volume of solution, and could provide dependable results when the volume was higher than 160 μL. Measurements of Staphylococcus aureus demonstrated detection limits of 104 copies μL−1 for genomic DNA nuc and 2.2 × 101 CFU mL−1 for milk cultured with 8 h. This method showed comparable sensitivity as the gel electrophoresis, avoided complicated operations and reduced the cost, demonstrating a promising potential to facilitate the detection of nucleic acid products.

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC-MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10 minutes. Calibration curves of all dNTPs ranged from 62.5–2,500 femtomole injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was

As an important epigenetic modification, 5-formylcytosine (5fC) plays a vital role in cell differentiation, genomic DNA structure and function regulation. Its low abundance in all suckler tissues and cells and high structural similarity with cytosine (C) and its derivatives, the sensitive and selective detection method for 5fC is required. In this work, a photoelectrochemical biosensor was constructed using BiIO4-WS2 composite as photosensitive material, the synergy of ternary heterojunction of BiIO4-WS2/CuO as signal amplification technology and 5-formylcytosine deoxyribonucleoside (5fdC) as target. Firstly, the ITO electrode modified by BiIO4-WS2 was aminated by (3- aminopropyl) triethoxysilane. 5fC was captured on the electrode surface with amidogen as recognition reagent, based on the amide bond formed between amidogen and aldehyde group. Then, 4-carboxyphenylboronic acid (4-CPBA) was linked by covalent reaction between boronic acid and O-diol of 5fdC. Finally, the signal amplification material of amino-functionalized CuO was deposited on the electrode surface through the formation of amide bond based on −COOH of 4-CPBA and –NH2 on CuO. In view of the constitution of a ternary heterojunction between CuO and BiIO4-WS2, the photoactivity of BiIO4-WS2 was greatly enhanced, which also increased the detection sensitivity with the wide examination area from 0.005 to 200 nM and low limit of detection was 0.38pM (3σ). Based on the specific covalent identification pattern, this method showed high selectivity for 5fC, even discriminating 5fC with C and 5hmC. The impacts of antibiotics and heavy metals on 5fdC content change in genomic DNA of wheat tissues were investigated, and thus the feasibility of this work was evaluated.

A new type of Forster Resonance Energy Transfer (FRET) system using highly emissive isomorphic nucleobase analogues is reported. The FRET pair consists of 2-aminothieno[3,4-d]pyrimidine G-mimic deoxyribonucleoside (th dG) as an energy donor and 1,3-diaza-2-oxophenothiazine (tC) as an energy acceptor. The distance and orientation between donor and acceptor was controlled by systematic incorporation of th dG and tC into DNA sequences to investigate the FRET efficiencies. This is the first Watson-Crick base-pairable FRET pair to produce vivid colors. In addition, this nucleic acid-based FRET pair was used to monitor DNA conformation and achieved visualization of the B-Z transition.

A grand challenge that crosses synthetic chemistry and biology is the scalable production of functional analogues of biomacromolecules. We have focused our attention on the use of deoxynucleoside building blocks bearing non-natural bases to develop a synthetic methodology that allows for the construction of high molecular weight deoxynucleotide polymers. Our six-membered cyclic phosphoester ring-opening polymerization strategy is demonstrated, herein, by an initial preparation of novel polyphosphoesters, comprised of butenyl-functionalized deoxyribonucleoside repeat units, connected via 3′,5′-backbone linkages. A thymidine-derived bicyclic monomer, 3′,5′-cyclic 3-(3-butenyl) thymidine ethylphosphate, was synthesized in two steps directly from thymidine, via butenylation and diastereoselective cyclization promoted by N,N-dimethyl-4-aminopyridine. Computational modeling of the six-membered 3′,5′-cyclic phosphoester ring derived from deoxyribose indicated strain energies at least 5.4 kcal/mol higher than tho...

We designed and synthesized a novel artificial 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2',4'-BNA/LNA with 5-methylcytosine or 2'-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2',4'-BNA/LNA moiety in DNA/RNA duplexes.

The authors report on a rapid voltammetric method for simultaneous determination of the pathogens E. coli and Salmonella typhimurium (S. typh.) by detecting the rfbE gene of E. coli O157:H7 and gyrB gene of S. typh., respectively, and by using polymerase-assisted target recycling amplification. The assay was constructed by self-assembly of the respective hairpin probes (labeled with the electrochemical probes Methylene Blue and ferrocene) on the surface of a gold electrode. After hybridization between target DNA and hairpin probes (HPs) has occurred, the primers hybridize with the open-chain HPs and initiate extension reactions in the presence of polymerase and deoxyribonucleoside triphosphates. This results in the release of the redox labels from the electrode surface and the target dissociating from the HPs. The released target will bind to other HPs to activate new cycles, which results in enhanced suppression of current, measured best at −0.27 V and +0.36 V (vs. Ag/AgCl) for parallel detection of E. coli DNA and S. typh. DNA, respectively. The method presented here based on target recycling amplification and its integration into multiplexed electrochemical detection of pathogens was successfully applied to quantitative determination of E. coli O157:H7 and S. typh. in synthetic samples. In our perception, the strategy presented here represents a rapid and universal platform for sensitive and multiplexed quantitation of pathogens and related molecular diagnostic targets of relevance in food safety control.

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10-13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.

(18)F-clofarabine, a nucleotide purine analog, is a substrate for deoxycytidine kinase (dCK), a key enzyme in the deoxyribonucleoside salvage pathway. (18)F-clofarabine might be used to measure dCK expression and thus serve as a predictive biomarker for tumor responses to dCK-dependent prodrugs or small-molecule dCK inhibitors, respectively. As a prerequisite for clinical translation, we determined the human whole-body and organ dosimetry of (18)F-clofarabine. Methods: Five healthy volunteers were injected intravenously with 232.4 ± 1.5 MBq of (18)F-clofarabine. Immediately after tracer injection, a dynamic scan of the entire chest was acquired for 30 min. This was followed by 3 static whole-body scans at 45, 90, and 135 min after tracer injection. Regions of interest were drawn around multiple organs on the CT scan and copied to the PET scans. Organ activity was determined and absorbed dose was estimated with OLINDA/EXM software. Results: The urinary bladder (critical organ), liver, kidney, and spleen exhibited the highest uptake. For an activity of 250 MBq, the absorbed doses in the bladder, liver, kidney, and spleen were 58.5, 6.6, 6.3, and 4.3 mGy, respectively. The average effective dose coefficient was 5.1 mSv. Conclusion: Our results hint that (18)F-clofarabine can be used safely in humans to measure tissue dCK expression. Future studies will determine whether (18)F-clofarabine may serve as a predictive biomarker for responses to dCK-dependent prodrugs or small-molecule dCK inhibitors.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.

A recombinant deoxyribonucleoside kinase from Drosophila melanogaster with a deletion of the last 20 amino acid residues (named DmdNKΔC20) was hypothesized as a potential therapeutic tool for gene therapy due to its broad substrate specificity and better catalytic efficiency towards nucleosides and nucleoside analogs. This study was designed to evaluate the effect of DmdNKΔC20 for sensitizing human cancer cell lines towards gemcitabine and to further investigate its role in reversal of acquired drug resistance in gemcitabine-resistant cancer cell line. The DmdNKΔC20 gene was delivered to three different cancer cell lines, including breast, colon and liver cancer cells, using lipid-mediated transfection reagent. After transfection, gene expression of DmdNKΔC20 was confirmed by reverse transcription quantitative PCR (qRT-PCR) and the combined effect of DmdNKΔC20 and gemcitabine based cytotoxicity was observed by cell viability assay. We further evolved a gemcitabine-resistant breast cancer cell line (named MCF7-R) through directed evolution in the laboratory, which showed 375-fold more resistance compared to parental MCF7 cells. Upon transfection with DmdNKΔC20 gene, MCF7-R cells showed 83-fold higher sensitivity to gemcitabine compared to the control group of MCF7-R cells. Moreover, we observed 79% higher expression of p21 protein in transfected MCF7-R cells, which may indicate induction of apoptosis. Our findings highlight the importance and therapeutic potential of DmdNKΔC20 in combined gene/chemotherapy approach to target a wide range of cancers, particularly gemcitabine-resistant cancers.