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1.
A target-driven DNA association was designed to initiate cyclic assembly of hairpins, which led to an enzyme-free amplification strategy for detection of a nucleic acid or aptamer substrate and flexible construction of logic gates. The cyclic system contained two ssDNA (S1 and S2) and two hairpins (H1 and H2). These ssDNA could co-recognize the target to produce an S1–target–S2 structure, which brought their toehold and branch-migration domains into close proximity to initiate the cyclic assembly of hairpins. The assembly product further induced the dissociation of a double-stranded probe DNA (Q:F) via toehold-mediated strand displacement to switch the fluorescence signal. This method could detect DNA and ATP as model analytes down to 21.6 pM and 38 nM, respectively. By designing different DNA input strands, the “AND”, “INHIBIT” and “NAND” logic gates could be activated to achieve the output signal. The proposed biosensing and logic gate operation platform showed potential applications in disease diagnosis. 相似文献
2.
Miao-Ping Chien Christopher A. Werley Samouil L. Farhi Adam E. Cohen 《Chemical science》2015,6(3):1701-1705
Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. 相似文献
3.
Non-SELEX selection of aptamers 总被引:5,自引:0,他引:5
Berezovski M Musheev M Drabovich A Krylov SN 《Journal of the American Chemical Society》2006,128(5):1410-1411
Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX. 相似文献
4.
Francesco V. Reddavide Weilin Lin Sarah Lehnert Dr. Yixin Zhang 《Angewandte Chemie (International ed. in English)》2015,54(27):7924-7928
Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA‐encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal‐to‐noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA‐encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high‐affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA‐templated chemical reactions. 相似文献
5.
Angus E. McMillan Wilson W. X. Wu Paula L. Nichols Benedikt M. Wanner Jeffrey W. Bode 《Chemical science》2022,13(48):14292
As a result of high false positive rates in virtual screening campaigns, prospective hits must be synthesised for validation. When done manually, this is a time consuming and laborious process. Large “on-demand” virtual libraries (>7 × 1012 members), suitable for preparation using capsule-based automated synthesis and commercial building blocks, were evaluated to determine their structural novelty. One sub-library, constructed from iSnAP capsules, aldehydes and amines, contains unique scaffolds with drug-like physicochemical properties. Virtual screening hits from this iSnAP library were prepared in an automated fashion for evaluation against Aedes aegypti and Phytophthora infestans. In comparison to manual workflows, this approach provided a 10-fold improvement in user efficiency. A streamlined method of relative stereochemical assignment was also devised to augment the rapid synthesis. User efficiency was further improved to 100-fold by downscaling and parallelising capsule-based chemistry on 96-well plates equipped with filter bases. This work demonstrates that automated synthesis consoles can enable the rapid and reliable preparation of attractive virtual screening hits from large virtual libraries.A compact and operationally simple automation technology can prepare virtual screening hits from a large on-demand library of drug-like molecules. 相似文献
6.
Yuqing Deng Jianzhao Peng Feng Xiong Yinan Song Yu Zhou Jianfu Zhang Fong Sang Lam Chao Xie Wenyin Shen Yiran Huang Ling Meng Xiaoyu Li 《Angewandte Chemie (International ed. in English)》2020,59(35):14965-14972
Dynamic combinatorial libraries (DCLs) is a powerful tool for ligand discovery in biomedical research; however, the application of DCLs has been hampered by their low diversity. Recently, the concept of DNA encoding has been employed in DCLs to create DNA‐encoded dynamic libraries (DEDLs); however, all current DEDLs are limited to fragment identification, and a challenging process of fragment linking is required after selection. We report an anchor‐directed DEDL approach that can identify full ligand structures from large‐scale DEDLs. This method is also able to convert unbiased libraries into focused ones targeting specific protein classes. We demonstrated this method by selecting DEDLs against five proteins, and novel inhibitors were identified for all targets. Notably, several selective BD1/BD2 inhibitors were identified from the selections against bromodomain 4 (BRD4), an important anti‐cancer drug target. This work may provide a broadly applicable method for inhibitor discovery. 相似文献
7.
Salt‐Induced Adaptation of a Dynamic Combinatorial Library of Pseudopeptidic Macrocycles: Unraveling the Electrostatic Effects in Mixed Aqueous Media
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Joan Atcher Dr. Alejandra Moure Dr. Jordi Bujons Dr. Ignacio Alfonso 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(18):6869-6878
Dynamic combinatorial libraries are powerful systems for studying adaptive behaviors and relationships, as models of more complex molecular networks. With this aim, we set up a chemically diverse dynamic library of pseudopeptidic macrocycles containing amino‐acid side chains with differently charged residues (negative, positive, and neutral). The responsive ability of this complex library upon the increase of the ionic strength has been thoroughly studied. The families of the macrocyclic members concentrating charges of the same sign showed a large increase in its proportion as the ionic strength increases, whereas those with residues of opposite charges showed the reverse behavior. This observation suggested an electrostatic shielding effect of the salt within the library of macrocycles. The top‐down deconvolution of the library allowed us to obtain the fundamental thermodynamic information connecting the library members (exchange equilibrium constants), as well as to parameterize the adaptation to the external stimulus. We also visualized the physicochemical driving forces for the process by structural analysis using NMR spectroscopy and molecular modeling. This knowledge permitted the full understanding of the whole dynamic library and also the de novo design of dynamic chemical systems with tailored co‐adaptive relationships, containing competing or cooperating species. This study highlights the utility of dynamic combinatorial libraries in the emerging field of systems chemistry. 相似文献
8.
Markus Leimbacher Dr. Yixin Zhang Dr. Luca Mannocci Michael Stravs Dr. Tim Geppert Dr. Jörg Scheuermann Prof. Gisbert Schneider Prof. Dario Neri 《Chemistry (Weinheim an der Bergstrasse, Germany)》2012,18(25):7729-7737
Libraries of chemical compounds individually coupled to encoding DNA tags (DNA‐encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high‐quality DNA‐encoded chemical library comprising 30 000 drug‐like compounds; this was screened in 170 different affinity capture experiments. High‐throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor‐associated antigen carbonic anhydrase IX (CA IX) and the pro‐inflammatory cytokine interleukin‐2 (IL‐2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL‐2 was confirmed by molecular docking. Our findings suggest that DNA‐encoded chemical libraries allow the facile identification of drug‐like ligands principally to any protein of choice, including molecules capable of disrupting high‐affinity protein–protein interactions. 相似文献
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Clickmers are chemically modified aptamers representing an innovative reagent class for developing binders for biomolecules with great impact on therapeutic and diagnostic applications. To establish a novel layer for screening various chemical entities, we developed a split–combine selection strategy simultaneously enriching for clickmers having different modifications. Due to the inherent design of this strategy, dynamic changes of DNA populations are traceable at an individual sequence level. Besides off-rate guided enrichment, the process makes the survival of the sequences most adapted to the applied selection condition observable. The underlying strategy provides unprecedented molecular insight into the selection process, based on which more sophisticated procedures will become pliable in the future.A split-combine selection approach reveals dynamic population changes in DNA libraries during in vitro selection procedures. 相似文献
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12.
A series of readily accessible, dynamic Diels–Alder reactions that are reversible at room temperature have been developed between anthracene derivatives as dienes and N‐phenyl‐1,2,4‐triazoline‐3,5‐dione as the dienophile. The adducts formed undergo reversible component exchange to form dynamic libraries of equilibrating cycloadducts. Furthermore, reversible adduct formation allows temperature‐dependent modulation of the fluorescent properties of anthracene components; a feature of potential interest for the design of optodynamic polymeric materials by careful selection and manipulation of these simple dienes and dienophiles. 相似文献
13.
Gobinda Das Bishnu P. Biswal Sharath Kandambeth V. Venkatesh Gagandeep Kaur Matthew Addicoat Thomas Heine Sandeep Verma Rahul Banerjee 《Chemical science》2015,6(7):3931-3939
Two new imide-based crystalline, porous, and chemically stable covalent organic frameworks (COFs) (TpBDH and TfpBDH) have been successfully synthesized employing solvothermal crystallization route. Furthermore, thin layered covalent organic nanosheets (CONs) were derived from these bulk COFs by the simple liquid phase exfoliation method. These 2D CONs showcase increased luminescence intensity compared to their bulk counterparts (COFs). Notably, TfpBDH-CONs showcase good selectivity and prominent, direct visual detection towards different nitroaromatic analytes over TpBDH-CONs. Quite interestingly, TfpBDH-CONs exhibit a superior “turn-on” detection capability for 2,4,6-trinitrophenol (TNP) in the solid state, but conversely, they also show a “turn-off” detection in the dispersion state. These findings describe a new approach towards developing an efficient, promising fluorescence chemosensor material for both visual and spectroscopic detection of nitroaromatic compounds with very low [10–5 (M)] analyte concentrations. 相似文献
14.
Researchers seeking to improve the efficiency and cost effectiveness of the bioactive small-molecule discovery process have recently embraced selection-based approaches, which in principle offer much higher throughput and simpler infrastructure requirements compared with traditional small-molecule screening methods. Since selection methods benefit greatly from an information-encoding molecule that can be readily amplified and decoded, several academic and industrial groups have turned to DNA as the basis for library encoding and, in some cases, library synthesis. The resulting DNA-encoded synthetic small-molecule libraries, integrated with the high sensitivity of PCR and the recent development of ultra high-throughput DNA sequencing technology, can be evaluated very rapidly for binding or bond formation with a target of interest while consuming minimal quantities of material and requiring only modest investments of time and equipment. In this tutorial review we describe the development of two classes of approaches for encoding chemical structures and reactivity with DNA: DNA-recorded library synthesis, in which encoding and library synthesis take place separately, and DNA-directed library synthesis, in which DNA both encodes and templates library synthesis. We also describe in vitro selection methods used to evaluate DNA-encoded libraries and summarize successful applications of these approaches to the discovery of bioactive small molecules and novel chemical reactivity. 相似文献
15.
Selection of smart aptamers by equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) 总被引:8,自引:0,他引:8
Drabovich A Berezovski M Krylov SN 《Journal of the American Chemical Society》2005,127(32):11224-11225
We coin a term of "smart aptamers", which describes aptamers with predefined binding parameters of their interaction with the target. Here, we introduce a method for selection of smart aptamers with predefined values of Kd: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Conceptually, a mixture of a target with a DNA (RNA) library is prepared and equilibrated. A plug of the equilibrium mixture is injected into a capillary prefilled with a run buffer containing the target at the concentration identical to the target concentration in the equilibrium mixture. The components of the equilibrium mixture are separated by capillary electrophoresis while equilibrium is maintained between the target and aptamers. The unique feature of ECEEM is that aptamers with different Kd values migrate with different and predictable mobilities. Thus, collecting fractions with different mobilities results in smart aptamers with different and predefined Kd values. In this proof-of-principle work, we used ECEEM to select smart aptamers for MutS protein, for which aptamers have never been previously selected. Three rounds of ECEEM-based selection were sufficient to obtain smart aptamers with Kd values approaching theoretically predicted ones. ECEEM is the first method for aptamer selection whose ability to generate smart aptamers has been experimentally proven. 相似文献
16.
Lei Li Yunpeng Liu Cheng Ma Jingyao Qu Angie D. Calderon Baolin Wu Na Wei Xuan Wang Yuxi Guo Zhongying Xiao Jing Song Go Sugiarto Yanhong Li Hai Yu Xi Chen Peng George Wang 《Chemical science》2015,6(10):5652-5661
Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to the complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycan (especially isomer) libraries is essential to help solve these tasks. We report herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-d-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of the enzymatic extension. Meanwhile, high performance liquid chromatography with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS2 and NMR. In summary, the CSEE strategy provides a practical approach for “mass production” of structurally defined N-glycans, which are important standards and probes for glycoscience. 相似文献
17.
Takayuki Miki Keigo Namii Kenta Seko Shota Kakehi Goshi Moro Hisakazu Mihara 《Chemical science》2022,13(43):12634
Phage display is the most widely used technique to discover de novo peptides that bind to target proteins. However, it is associated with some challenges such as compositional bias. In this study, to overcome these difficulties, we devised a ‘pattern enrichment analysis.’ In this method, two samples (one obtained by affinity selection, the other simply amplified without selection) are prepared, and the two sequence datasets read on next-generation sequencer are compared to find the three-residue pattern most enriched in the selected sample. This allows us to compare two sequence datasets with high coverage and facilitates the identification of peptide sequences and the key residues for binding. We also demonstrated that this approach in the combination with structured peptide libraries allowed spatial mapping of the enriched sequence patterns. Here, we prepared a phage library displaying chemically stapled helical peptides with the X1C2X3X4X5X6X7X8C9X10 sequence, where X is any amino acid. To validate our method, we performed screening against the HDM2 protein. The results showed that the hydrophobic residues (Phe, Tyr, Trp and Leu) that are key to interactions with HDM2 were clearly identified by the pattern enrichment analysis. We also performed selection targeting the SARS-CoV-2 spike RBD in the same manner. The results showed that similar patterns were enriched among the hit peptides that inhibited the protein–protein interaction.To effectively identify helical peptide binders from a phage library, we developed “pattern enrichment analysis,” which finds the enriched three-residue patterns by comparison with a control sample. 相似文献
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19.
Luciana Lisa Lao Dr. Jean‐Louis Schmitt Dr. Jean‐Marie Lehn Prof. Dr. 《Chemistry (Weinheim an der Bergstrasse, Germany)》2010,16(16):4903-4910
Conversion of macrocyclic imine entities into helical strands was achieved through three‐ and four‐component exchange reactions within constitutionally dynamic libraries. The generation of sequences of the intrinsic helicity codon, based on the hydrazone–pyrimidine fragment obtained by condensation of pyrimidine dialdehyde A with pyrimidine bis‐hydrazine B , shifted the equilibrium between all the possible macrocycles and strands towards the full expression (>98%) of helical product [ A / B ]. Furthermore, it was shown that chain folding accelerated the dynamic exchange reactions among the library members. Lastly, in four‐component experiments (involving A , B , E and either C or D ), even though the macrocyclic entities ([ A / C ], [ B / E ]; [ A / D ], [ B / E ]) were the kinetically preferred products, over time dialdehyde A relinquished its initial diamine partners C or D to opt for bis‐hydrazine B , which allowed the preferential formation of the helically folded strand. The present results indicate that self‐organisation pressure was able to drive the dynamic system towards the selective generation of the strand undergoing helical folding. 相似文献
20.
Ramström O Lohmann S Bunyapaiboonsri T Lehn JM 《Chemistry (Weinheim an der Bergstrasse, Germany)》2004,10(7):1711-1715
Dynamic combinatorial chemistry (DCC) has emerged as an efficient approach to receptor/ligand identification based on the generation of combinatorial libraries by reversible interconversion of the library constituents. In this study, the implementation of such libraries on carbohydrate-lectin interactions was examined with the plant lectin Concanavalin A as a target species. Dynamic carbohydrate libraries were generated from a pool of carbohydrate aldehydes and hydrazide linker/scaffold components through reversible acylhydrazone exchange, resulting in libraries containing up to 474 constituents. Dynamic deconvolution allowed the efficient identification of the structural features required for binding to Concanavalin A and the selection of a strong binder, a tritopic mannoside, showing an IC(50)-value of 22 microM. 相似文献