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1.
Advancing Small‐Molecule‐Based Chemical Biology with Next‐Generation Sequencing Technologies 下载免费PDF全文
Chandran Anandhakumar Seiichiro Kizaki Dr. Toshikazu Bando Dr. Ganesh N. Pandian Prof. Dr. Hiroshi Sugiyama 《Chembiochem : a European journal of chemical biology》2015,16(1):20-38
Next‐generation‐sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA‐sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in‐depth insight into small‐molecule‐triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small‐molecule development and screening. 相似文献
2.
Identification of Sequence Specificity of 5‐Methylcytosine Oxidation by Tet1 Protein with High‐Throughput Sequencing 下载免费PDF全文
Seiichiro Kizaki Dr. Anandhakumar Chandran Prof. Dr. Hiroshi Sugiyama 《Chembiochem : a European journal of chemical biology》2016,17(5):403-406
Tet (ten‐eleven translocation) family proteins have the ability to oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic‐level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high‐throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×104‐member mC‐containing random DNA‐sequence library was constructed. The library was subjected to Tet‐reactive pulldown followed by high‐throughput sequencing. Analysis of the obtained sequence data identified the Tet1‐reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. 相似文献
3.
Hao Li Raphael M. Franzini Christopher Bruner Prof. Dr. Eric T. Kool 《Chembiochem : a European journal of chemical biology》2010,11(15):2132-2137
We describe the development of templated fluorogenic chemistry for detection of specific sequences of duplex DNA in solution. In this approach, two modified homopyrimidine oligodeoxynucleotide probes are designed to bind by triple‐helix formation at adjacent positions on a specific purine‐rich target sequence of duplex DNA. One fluorescein‐labeled probe contains an α‐azidoether linker to a fluorescence quencher; the second (trigger) probe carries a triarylphosphine group that is designed to reduce the azide and cleave the linker. The data showed that at pH 5.6 these probes yielded a strong fluorescence signal within minutes on addition to a complementary homopurine duplex DNA target. The signal increased by a factor of about 60, and was completely dependent on the presence of the target DNA. Replacement of cytosine in the probes with pseudoisocytosine allowed the templated chemistry to proceed readily at pH 7. Single nucleotide mismatches in the target oligonucleotide slowed the templated reaction considerably; this demonstrated high sequence selectivity. The use of templated fluorogenic chemistry for detection of duplex DNAs has not been previously reported and could allow detection of double‐stranded DNA, at least for homopurine–homopyrimidine target sites, under native and nondenaturing conditions. 相似文献
4.
Raphael M. Franzini Eric T. Kool Prof. 《Chembiochem : a European journal of chemical biology》2008,9(18):2981-2988
Templated nucleic acid detection is an emerging bioanalytical method that makes use of the target DNA or RNA strand to initiate a fluorogenic reaction. The Staudinger reduction holds particular promise for templated sensing of nucleic acids because the involved functional groups are highly chemoselective. Here, the azidomethoxy group, which can be removed under Staudinger conditions, is used to cage 7‐hydroxycoumarin fluorophores. Reduction by phosphines and subsequent loss of the azidomethoxy substituent induce a significant bathochromic shift of the major absorbance band in the near UV region. When excited at the appropriate wavelength, this change in the absorbance spectrum translates into a substantial fluorescence turn‐on signal. The described profluorophores are readily conjugated to amino‐modified DNAs and are rapidly uncaged by a triphenylphosphine–DNA probe under the control of a DNA template. In addition, turnover of the probes on the target strand occurs and yields substantial signal amplification. 相似文献
5.
Highly Efficient Fluorescent Interstrand Photo‐crosslinking of DNA Duplexes Labeled with 5‐Fluoro‐4‐thio‐2′‐O‐methyluridine 下载免费PDF全文
Dr. Joanna Nowak‐Karnowska Ziad Chebib Prof. Jan Milecki Prof. Stefan Franzen Prof. Bohdan Skalski 《Chembiochem : a European journal of chemical biology》2014,15(14):2045-2049
The formation of a fluorescent photoadduct between 5‐fluoro‐4‐thiouridine ( FS U ), in the sequence context 5′‐A FS U A‐3′ and incorporated into a synthetic oligonucleotide either at its 3′‐ or 5′‐end, and one of the thymines of the TAT motif in a complementary target DNA strand led to photo‐crosslinking of the two strands for several oligonucleotide constructs. Enzymatic digestion, MS, UV, and fluorescence spectral analyses of the interstrand crosslinked oligonucleotides revealed the identity of the thymidine that participates in the photo‐crosslinking reaction as well as the diastereomeric structures of the crosslinks. The proposed pathways of interstrand photo‐crosslinking are supported by experiments with isotopically labeled oligonucleotide constructs and visualized by means of molecular dynamics simulations. 相似文献
6.
Next‐Generation Sequencing Studies Guide the Design of Pyrrole‐Imidazole Polyamides with Improved Binding Specificity by the Addition of β‐Alanine 下载免费PDF全文
Chandran Anandhakumar Yue Li Seiichiro Kizaki Dr. Ganesh N. Pandian Kaori Hashiya Dr. Toshikazu Bando Prof. Hiroshi Sugiyama 《Chembiochem : a European journal of chemical biology》2014,15(18):2647-2651
7.
Luca Mannocci Dr. Alessandra Villa Roberto Sommavilla Yixin Zhang Markus G. Grütter Nadine Keller Lutz Jermutus Ronald H. Jackson Jörg Scheuermann Dario Neri Prof. Dr. 《ChemMedChem》2010,5(4):584-590
Bcl‐xL is an antiapoptotic member of the Bcl‐2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl‐xL from a DNA‐encoded chemical library using affinity‐capture selections and massively parallel high‐throughput sequencing of >30 000 sequence tags of library members. The most potent binder identified, compound 19 / 93 [(R)‐3‐(amido indomethacin)‐4‐(naphthalen‐1‐yl)butanoic acid], bound to Bcl‐xL with a dissociation constant (Kd) of 930 nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function. 相似文献
8.
Samwel K. Cheruiyot Dr. Eriks Rozners 《Chembiochem : a European journal of chemical biology》2016,17(16):1558-1562
Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex‐forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2‐aminopyridine (M) nucleobase to a G‐C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3‐(1‐phenylethynyl)‐M and phenylpyrrolo‐M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple‐helix formation with cognate double‐stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3‐(1‐phenylethynyl)‐M may be used as a fluorescent nucleobase to study PNA–RNA triple‐helix formation. 相似文献
9.
Dr. Nina Svensen Prof. Olve B. Peersen Prof. Samie R. Jaffrey 《Chembiochem : a European journal of chemical biology》2016,17(17):1628-1635
Methods for displaying large numbers of peptides on solid surfaces are essential for high‐throughput characterization of peptide function and binding properties. Here we describe a method for converting the >107 flow cell‐bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow‐cell‐bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3Dpol. The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein‐ or small molecule‐binding aptamers. We used the mRNA‐display approach to synthesize flow‐cell‐tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide‐based assays. 相似文献
10.
Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells 下载免费PDF全文
Iuliia A. Karpenko Rémy Kreder Christel Valencia Dr. Pascal Villa Dr. Christiane Mendre Dr. Bernard Mouillac Prof. Dr. Yves Mély Prof. Dr. Marcel Hibert Dr. Dominique Bonnet Dr. Andrey S. Klymchenko 《Chembiochem : a European journal of chemical biology》2014,15(3):359-363
Classical fluorescence‐based approaches to monitor ligand–protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn‐on probes for a G protein‐coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor‐specific turn‐on response. The new ligand was successfully applied for background‐free imaging and quantification of oxytocin receptors in living cells. 相似文献
11.
Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement 下载免费PDF全文
Dr. Wouter W. Kallemeijn Saskia Scheij Tineke M. Voorn‐Brouwer Dr. Martin D. Witte Marri Verhoek Prof. Dr. Hermen S. Overkleeft Dr. Rolf G. Boot Prof. Dr. Johannes M. F. G. Aerts 《Chembiochem : a European journal of chemical biology》2016,17(18):1698-1704
β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms. 相似文献
12.
Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase 下载免费PDF全文
Eiman A. Osman Dr. Tendai Gadzikwa Dr. Julianne M. Gibbs 《Chembiochem : a European journal of chemical biology》2018,19(19):2081-2087
The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a kobs value of 1.1 min?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches. 相似文献
13.
Mechanism‐Based Inhibitor of DNA Cytosine‐5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2‐Amino‐4‐Halopyridine‐C‐Nucleoside 下载免费PDF全文
Dr. Kousuke Sato Yuma Kunitomo Yukiko Kasai Shohei Utsumi Prof. Isao Suetake Prof. Shoji Tajima Prof. Satoshi Ichikawa Prof. Akira Matsuda 《Chembiochem : a European journal of chemical biology》2018,19(8):865-872
In chromatin, 5‐methylcytosine (mC), which represents the fifth nucleobase in genomic DNA, plays a role as an inducer of epigenetic changes. Tumor cells exhibit aberrant DNA methylation patterns, and inhibition of human DNA cytosine‐5 methyltransferase (DNMT), which is responsible for generating mC in CpG sequences, is an effective strategy to treat various cancers. Here, we describe the design, synthesis, and evaluation of the properties of 2‐amino‐4‐halopyridine‐C‐nucleosides (dXP) and oligodeoxyribonucleotides (ODNs) containing dXP as a novel mechanism‐based inhibitor of DNMTs. The designed ODN containing XPpG forms a complex with DNMTs by covalent bonding through a nucleophilic aromatic substitution (SNAr) reaction, and its cell proliferation activity is investigated. This study suggests that dXP in a CpG sequence of DNA could serve as a potential nucleic acid drug lead in cancer chemotherapy and a useful chemical probe for studies of epigenetics. Our molecular design using a SNAr reaction would be useful for DNMTs and other protein–DNA interactions. 相似文献
14.
1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA 下载免费PDF全文
Krisana Peewasan Prof. Dr. Hans‐Achim Wagenknecht 《Chembiochem : a European journal of chemical biology》2017,18(15):1473-1476
In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand. 相似文献
15.
Sarah K. Edwards Dr. Toshikazu Ono Dr. Shenliang Wang Dr. Wei Jiang Dr. Raphael M. Franzini Dr. Jong Wha Jung Ke Min Chan Prof. Eric T. Kool 《Chembiochem : a European journal of chemical biology》2015,16(11):1637-1646
The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays. 相似文献
16.
Toshihide Takeuchi Dr. Valentina Bagnacani Francesco Sansone Prof. Stefan Matile Prof. 《Chembiochem : a European journal of chemical biology》2009,10(17):2793-2799
We report that amphiphilic counterions can enable DNA to act as cation carrier, enzyme detector and biosensor. Calf thymus DNA is used as example throughout the study. Evaluation of a series of counterion activators suggests that strong amphiphilicity, alkyl or calix[4]arene tails and guanidinium cations give best results, whereas weak amphiphilicity, bola‐amphiphilicity, planar aryl tails and ammonium cations are less satisfactory for various reasons. In the U‐tube, DNA–counterion complexes can carry cations such as safranin O or p‐xylene‐bis‐pyridinium bromide (DPX) across bulk chloroform membranes, whereas anions such as carboxyfluorescein (CF) and (8‐Hydroxy‐1,3,6‐pyrenetrisulfonate (HPTS) are not transported. Uptake of DNA–counterion complexes into intact vesicles is demonstrated by DNA trapping experiments with internal polylysine. Comparison of results from different assays suggests that DNA–counterion complexes act as cation carriers under mild conditions, whereas pore formation and lysis dominate at higher concentrations. Applicability of DNA–counterion transporters for the detection of enzyme activity is demonstrated with phytate as an inactivating substrate and phytase as a reactivating enzyme. Compatibility with biosensing is exemplified with the fluorometric monitoring of phytate levels in almond extracts. The conceptual significance of these findings is briefly discussed, as are promising perspectives such as the application of DNA chemistry to multianalyte sensing in fluorogenic vesicles. 相似文献
17.
Gaetano Giammona Gennara Cavallaro Giovanna Pitarresi Elisa Pedone 《Polymer International》2000,49(1):93-98
In the present study the derivatization of two water‐soluble synthetic polymers, α,β‐poly(N‐2‐hydroxyethyl)‐DL ‐aspartamide (PHEA) and α,β‐polyasparthylhydrazide (PAHy), with glycidyltrimethylammonium chloride (GTA) is described. This reaction permits the introduction of positive charges in the macromolecular chains of PHEA and PAHy in order to make easier the electrostatic interaction with DNA. Different parameters affect the reaction of derivatization, such as GTA concentration and reaction time. PHEA reacts partially and slowly with GTA; on the contrary the reaction of PAHy with GTA is more rapid and extensive. The derivatization of PHEA and PAHy with GTA is a convenient method to introduce positive groups in their chains and it permits the preparation of interpolyelectrolyte complexes with DNA. © 2000 Society of Chemical Industry 相似文献
18.
The DNA adsorption properties of poly‐L ‐lysine‐immobilized poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane were investigated. The pHEMA membrane was prepared by UV‐initiated photopolymerization and activated with epichlorohydrin. Poly‐L ‐lysine was then immobilized on the activated pHEMA membrane by covalent bonding, via a direct chemical reaction between the amino group of poly‐L ‐lysine and the epoxy group of pHEMA. The poly‐L ‐lysine content of the membrane was determined as 1537 mg m?2. The poly‐L ‐lysine‐immobilized membrane was utilized as an adsorbent in DNA adsorption experiments. The maximum adsorption of DNA on the poly‐L ‐lysine‐immobilized pHEMA membrane was observed at 4 °C from phosphate‐buffered salt solution (pH 7.4, 0.1 M; NaCl 0.5 M) containing different amounts of DNA. The non‐specific adsorption of DNA on the plain pHEMA membrane was low (about 263 mg m?2). Higher DNA adsorption values (up to 5849 mg m?2) were obtained in which the poly‐L ‐lysine‐immobilized pHEMA membrane was used. Copyright © 2003 Society of Chemical Industry 相似文献
19.
NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real‐Time in vivo Monitoring of Drug Release 下载免费PDF全文
Orit Redy‐Keisar Shiran Ferber Prof. Ronit Satchi‐Fainaro Prof. Doron Shabat 《ChemMedChem》2015,10(6):999-1007
The ability to monitor drug release in vivo provides essential pharmacological information. We developed a new modular approach for the preparation of theranostic prodrugs with a turn‐ON near‐infrared (NIR) fluorescence mode of action. The prodrugs release their chemotherapeutic cargo and an active cyanine fluorophore upon reaction with a specific analyte. The prodrug platform is based on the fluorogenic dye QCy7; upon removal of a triggering substrate, the dye fluoresces, and the free drug is released. The evaluated camptothecin prodrug was activated by endogenous hydrogen peroxide produced in tumor cells in vitro and in vivo. Drug release and in vitro cytotoxicity were correlated with the emitted fluorescence. The prodrug activation was effectively imaged in real time in mice bearing tumors. The modular design of the QCy7 fluorogenic platform should allow the preparation of numerous other prodrugs with various triggering substrates and chemotherapeutic agents. We anticipate that the development of real‐time in vivo monitoring tools such as that described herein will pave the way for personalized therapy. 相似文献