Discovery of Small‐Molecule Interleukin‐2 Inhibitors from a DNA‐Encoded Chemical Library

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.     

Selection of DNA‐Encoded Dynamic Chemical Libraries for Direct Inhibitor Discovery

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.     

DNA‐Encoded Dynamic Combinatorial Chemical Libraries

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.     

Identification of Structure–Activity Relationships from Screening a Structurally Compact DNA‐Encoded Chemical Library

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA‐encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL‐100K, a DNA‐encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA‐sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate‐specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small‐molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.     

A DNA‐Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Here we show a seven‐step chemical synthesis of a DNA‐encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide‐polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA‐encoded libraries, while revealing areas where new DNA synthetic methods are needed.     

RASS‐Enabled S/P−C and S−N Bond Formation for DEL Synthesis

DNA encoded libraries (DEL) have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently reversible adsorption to solid support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on‐DNA chemistries to incorporate medicinally relevant and C?S, C?P and N?S linkages into DELs, which are underrepresented in the canonical methods.     

Hybridization Detection of Osmium Tetroxide Bipyridine‐Labeled DNA and RNA on Heated Gold Wire Electrodes

We report about hybridization detection of different nucleic acids on capture probe‐modified heated gold wire electrodes. We have compared three kinds of nucleic acid targets: DNA, uracil‐conjugated DNA, and RNA. All three sorts of nucleic acids targets could be labeled with osmium tetroxide bipyridine, hybridized with immobilized DNA capture probes and then detected by square‐wave voltammetry. Heating the gold electrode instead of the entire bulk hybridization solution leads to improved hybridization efficiency in most cases. The reason could be found in a thermal micro‐stirring effect around the heated wire electrode. Also selectivity was improved. Mismatches could be discriminated for DNA and uracil‐conjugated DNA targets. Mismatches in RNA strands, however, are more difficult to detect due to relatively stable secondary structures.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

High Sensitive Electrochemical Detection of Sequence‐Specific DNA Using Low Current Voltammetry

In this article, we report on efforts to construct a high sensitive electrochemical sensor with immobilized sandwich‐type DNA borne ferrocene (Fc) head for sequence‐specific DNA detection using ultramicroelectrode and low current voltammetry. Based on the difference in deformability between the bending rigid complementary DNA double helix and its anomalous flexile mismatches, the fully complementary target can be distinguished from mismatched targets including the single‐base mismatched target. Detection limit estimated as the amount of DNA is observed to be 100 fM via low current voltammetry. The method offers great promise of high sensitivity and selectivity simultaneously for effective gene identification.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

Sequence‐specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid‐DNA) in capillary and microfluidic electrophoretic separations

Reversible noncovalent but sequence‐dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite‐modified oligonucleotides has enabled sequence‐specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self‐assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid‐DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence‐dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information‐processing systems.     

Screening of Three Transition Metal‐Mediated Reactions Compatible with DNA‐Encoded Chemical Libraries

The construction of DNA‐encoded chemical libraries (DECLs) crucially relies on the availability of chemical reactions, which are DNA‐compatible and which exhibit high conversion rates for a large number of diverse substrates. In this work, we present our optimization and validation procedures for three copper and palladium‐catalyzed reactions (Suzuki cross‐coupling, Sonogashira cross‐coupling, and copper(I)‐catalyzed alkyne‐azide cycloaddition (CuAAC)), which have been successfully used by our group for the construction of large encoded libraries.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution‐phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence‐activated cell sorting (FACS) to individually measure the relative affinities of >10⁸ aptamer particles and sort them in a high‐throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high‐affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high‐quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.     

Small Targeted Cytotoxics: Current State and Promises from DNA‐Encoded Chemical Libraries

The targeted delivery of potent cytotoxic agents has emerged as a promising strategy for the treatment of cancer and other serious conditions. Traditionally, antibodies against markers of disease have been used as drug‐delivery vehicles. More recently, lower molecular weight ligands have been proposed for the generation of a novel class of targeted cytotoxics with improved properties. Advances in this field crucially rely on efficient methods for the identification and optimization of organic molecules capable of high‐affinity binding and selective recognition of target proteins. The advent of DNA‐encoded chemical libraries allows the construction and screening of compound collections of unprecedented size. In this Review, we survey developments in the field of small ligand‐based targeted cytotoxics and show how innovative library technologies will help develop the drugs of the future.     

Electrochemical Biosensors Based on Layer‐by‐Layer Assemblies

Layer‐by‐layer (LBL) assemblies, which have undergone great progress in the past decades, have been used widely in the construction of electrochemical biosensors. The LBL assemblies provide a strategy to rationally design the properties of immobilized films and enhance the performance of biosensors. The following review focuses on the application of LBL assembly technique on electrochemical enzyme biosensors, immunosensors and DNA sensors.     

Template‐Free Galvanic Nanostructuring of Gold Electrodes for Sensitive Electrochemical Biosensors

Gold nanostructuring of smooth gold disk‐electrodes has been performed by electrodeposition. FE‐SEM images indicate a strongly enlarged electrochemically active surface of the modified electrodes. This could be approved by the gold oxide reduction peak in cyclic voltammetry. The electrodes were also applied for sequence‐specific DNA detection. For this, complementary single stranded capture probes were immobilized on the electrodes and consecutively hybridized with complementary and noncomplementary [OsO₄(bipy)]‐labeled DNA targets. We found that intermediate dehybridization steps did not decrease the sensitivity for the specific target. Moreover, the surface modification increased the voltammetric signal by an approx. factor of 9. Further, an enhanced linear calibration range was observed. The presented simple nanostructuring process holds great promise for many electrochemical sensor applications.     

Determination of Tetracycline at a UV‐Irradiated DNA Film Modified Glassy Carbon Electrode

In this study, interaction of tetracycline (TC) and DNA in the Britton? Robinson buffer solution (BR) was studied by cyclic voltammetry. The experimental results reveal that TC can bind strongly to DNA and the association constant and binding number between TC and DNA was obtained. Then DNA was immobilized on a glassy carbon electrode by UV‐irradiation. Through this process, water‐soluble DNA was converted into insoluble materials, and a stable DNA film was formed on the electrode. The electrochemical oxidation behavior of TC was studied at UV‐irradiated DNA film modified glassy carbon electrode (UV‐DNA‐GCE). The response of modified electrode was optimized with respect to pH, accumulation time, ionic strength, drug concentration and other variables. TC at the surface of modified electrode showed a linear dynamic range of 0.30–90.00 µM and a detection limit of 0.27 µM. To demonstrate the applicability of the modified electrode, it was used for the analysis of real samples such as pharmaceutical formulations and milk.     

Site‐Directed,On‐Surface Assembly of DNA Nanostructures

Two‐dimensional DNA lattices have been assembled from DNA double‐crossover (DX) motifs on DNA‐encoded surfaces in a site‐specific manner. The lattices contained two types of single‐stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface‐bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA‐tethered probe molecules on the opposite side of the lattices exposed to the solution. Site‐specific lattice assembly and attachment of fluorophore‐labeled oligonucleotides and DNA–protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications.     

GenoMass ‐ a computer software for automated identification of oligonucleotide DNA adducts from LC‐MS analysis of DNA digests

In the investigation of oligonucleotides, DNA and their adducts by LC‐MS, a myriad of data are generated that make manual data processing quite difficult. This paper describes a ‘reversed pseudo‐combinatorial’ approach for fragment identification and the software implementation of this approach. Combinatorial isomer libraries are generated in silico to represent the digestion products of oligonucleotides, DNA or DNA adducts of various sizes. The software automatically calculates ion masses of each isomeric segment of the library, searches for them in complicated LC‐MS data, lists their intensities and plots extracted ion chromatograms (EIC). This customized new data analysis tool has enabled a study of the enzymatic behavior of a nuclease system in the digestion of normal and adducted DNA, and in the recognition of oligomers containing a carcinogen bound to a nucleobase. The software program potentially can be further expanded to postulate unknown DNA sequences and recognize the adduction sites. Copyright © 2008 John Wiley & Sons, Ltd.