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DNA-encoded library (DEL) technology is a powerful platform for the efficient identification of novel chemical matter in the early drug discovery process enabled by parallel screening of vast libraries of encoded small molecules through affinity selection and deep sequencing. While DEL selections provide rich data sets for computational drug discovery, the underlying technical factors influencing DEL data remain incompletely understood. This review systematically examines the key parameters affecting the chemical information in DEL data and their impact on hit triaging and machine learning integration. The need for rigorous data handling and interpretation is emphasized, with standardized methods being critical for the success of DEL-based approaches. Major challenges include the relationship between sequence counts and binding affinities, frequent hitters, and the influence of factors such as inhomogeneous library composition, DNA damage, and linkers on binding modes. Experimental artifacts, such as those caused by protein immobilization and screening matrix effects, further complicate data interpretation. Recent advancements in using machine learning to denoise DEL data and predict drug candidates are highlighted. This review offers practical guidance on adopting best practices for integrating robust methodologies, comprehensive data analysis, and computational tools to improve the accuracy and efficacy of DEL-driven hit discovery.

Sirtuin 6 (Sirt6), an NAD + -dependent deacylase, has emerged as a promising target for aging-related diseases and cancer. Advancing the medicinal chemistry of Sirt6 modulators is crucial for the development of chemical probes aimed at unraveling the intricate biological functions of Sirt6 and unlocking its therapeutic potential. A proprietary DNA-encoded library yielded Sirt6 inhibitor 2-Pr, displaying remarkable inhibitory activity and isoform-selectivity, and featuring a chemical structure distinct from reported Sirt6 modulators. In this study, we explore the inhibitory mechanism of 2-Pr, evaluating the impact of chemical modifications and presenting a crystal structure of the Sirt6/ADP-ribose/2-Pr complex. Notably, co-crystal structure analysis reveals an unexpected and unprecedented binding mode of Sirt6, with 2-Pr spanning the acyl channel of the enzyme, extending into the acetyl-lysine binding pocket, and reaching toward the C-site. This unique binding mode guides potential avenues for developing potent and selective Sirt6 inhibitors., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Chemoselective pairs of bioorthogonal reactants enable the simultaneous labeling of several biomolecules. Here, we access orthogonal click reactions by exploiting differences in frontier molecular orbital interaction energies in transition states. We establish that five-membered cyclic dienes are inert to isonitriles but readily react with strained alkynes, while tetrazines with bulky substituents readily react with isonitriles. Strained alkynes show an opposite reactivity pattern. The approach was demonstrated by orthogonally labeling two proteins with different fluorophores.

DNA-encoded libraries (DELs) are widely used in the discovery of drug candidates, and understanding their design principles is critical for accessing better libraries. Most DELs are combinatorial in nature and are synthesized by assembling sets of building blocks in specific topologies. In this study, different aspects of library topology were explored and their effect on DEL properties and chemical diversity was analyzed. We introduce a descriptor for DEL topological assignment (DELTA) and use it to examine the landscape of possible DEL topologies and their coverage in the literature. A generative topographic mapping analysis revealed that the impact of library topology on chemical space coverage is secondary to building block selection. Furthermore, it became apparent that the descriptor used to analyze chemical space dictates how structures cluster, with the effects of topology being apparent when using three-dimensional descriptors but not with common two-dimensional descriptors. This outcome points to potential challenges of attempts to predict DEL productivity based on chemical space analyses alone. While topology is rather inconsequential for defining the chemical space of encoded compounds, it greatly affects possible interactions with target proteins as illustrated in docking studies using NAD/NADP binding proteins as model receptors.

DNA-encoded chemical libraries (DECLs) interrogate the interactions of a target of interest with vast numbers of molecules. DECLs hence provide abundant information about the chemical ligand space for therapeutic targets, and there is considerable interest in methods for exploiting DECL screening data to predict novel ligands. Here we introduce one such approach and demonstrate its feasibility using the cancer-related poly-(ADP-ribose)transferase tankyrase 1 (TNKS1) as a model target. First, DECL affinity selections resulted in structurally diverse TNKS1 inhibitors with high potency including compound 2 with an IC 50 value of 0.8 nM. Additionally, TNKS1 hits from four DECLs were translated into pharmacophore models, which were exploited in combination with docking-based screening to identify TNKS1 ligand candidates in databases of commercially available compounds. This computational strategy afforded TNKS1 inhibitors that are outside the chemical space covered by the DECLs and yielded the drug-like lead compound 12 with an IC 50 value of 22 nM. The study further provided insights in the reliability of screening data and the effect of library design on hit compounds. In particular, the study revealed that while in general DECL screening data are in good agreement with off-DNA ligand binding, unpredictable interactions of the DNA-attachment linker with the target protein contribute to the noise in the affinity selection data., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Raphael Franzini reports financial support was provided by National Institutes of Health. Raphael Franzini reports financial support was provided by ALSAM Foundation. Herwig Schuler reports financial support was provided by Swedish Cancer Society. Dmitri Kireev reports financial support was provided by National Institutes of Health., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Reactions that occur under physiological conditions find diverse uses in the chemical and biological sciences. However, the limitations that biological systems place on chemical reactions restrict the number of such bioorthogonal reactions. A profound understanding of the mechanistic principles and structure-reactivity trends of these transformations is therefore critical to access new and improved versions of bioorthogonal chemistry. The present article reviews the mechanisms and substituent effects of some of the principal metal-free bioorthogonal reactions based on inverse-electron demand Diels-Alder reactions, 1,3-dipolar cycloadditions, and the Staudinger reaction. Mechanisms of modified versions that link these reactions to a dissociative step are further discussed. The presented summary is anticipated to aid the advancement of bioorthogonal chemistry.

Two critical steps in drug development are 1) the discovery of molecules that have the desired effects on a target, and 2) the optimization of such molecules into lead compounds with the required potency and pharmacokinetic properties for translation. DNA-encoded chemical libraries (DECLs) can nowadays yield hits with unprecedented ease, and lead-optimization is becoming the limiting step. Here we integrate DECL screening with structure-based computational methods to streamline the development of lead compounds. The presented workflow consists of enumerating a virtual combinatorial library (VCL) derived from a DECL screening hit and using computational binding prediction to identify molecules with enhanced properties relative to the original DECL hit. As proof-of-concept demonstration, we applied this approach to identify an inhibitor of PARP10 that is more potent and druglike than the original DECL screening hit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The isocyano group is a valuable functionality for bioorthogonal reactions because it rapidly reacts with tetrazines to either form stable conjugates or release payloads from 3-isocyanopropyl groups. Here we provide mechanistic insights into the dissociative steps that follow the initial cycloaddition and analyze how structural modifications affect these processes. Three main outcomes of this study have important implications for designing such groups for bioorthogonal applications. First, anion-stabilizing substituents at C-2 of the 3-isocyanopropyl group promote β-elimination and accelerate deprotection. Second, tetrazines with bulky substituents form stable imine conjugates even with primary isonitriles that are otherwise rapidly hydrolyzed. Third, the elimination step is independent from hydrolysis to the aldehyde and instead can occur directly from the imine intermediate. These findings will allow tuning the structures of tetrazine and isonitrile reactants for application in bioorthogonal ligation and release chemistry.

In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions., (This journal is © The Royal Society of Chemistry 2020.)

Bioorthogonal reactions that proceed readily under physiological conditions without interference from biomolecules have found widespread application in the life sciences. Complementary to the bioorthogonal reactions that ligate two molecules, reactions that release a molecule or cleave a linker are increasingly attracting interest. Such dissociative bioorthogonal reactions have a broad spectrum of uses, for example, in controlling bio-macromolecule activity, in drug delivery, and in diagnostic assays. This review article summarizes the developed bioorthogonal reactions linked to a release step, outlines representative areas of the applications of such reactions, and discusses aspects that require further improvement., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure-activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol -1  s -1 were accessible by combining bulky and electron-withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded chemical libraries are increasingly used in pharmaceutical research because they enable the rapid discovery of synthetic protein ligands. Here we explored whether target-class focused DNA-encoded chemical libraries can be cost-effective tools to achieve robust screening productivity for a series of proteins. The study revealed that a DNA-encoded library designed for NAD + -binding pockets (NADEL) effectively sampled the chemical binder space of enzymes with ADP-ribosyltransferase activity. The extracted information directed the synthesis of inhibitors for several enzymes including PARP15 and SIRT6. The high dissimilarity of NADEL screening fingerprints for different proteins translated into inhibitors that showed selectivity for their target. The discovery of patterns of enriched structures for six out of eight tested proteins is remarkable for a library of 58 302 DNA-tagged structures and illustrates the prospect of focused DNA-encoded libraries as economic alternatives to large library platforms.

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopropyl derivatives react with tetrazines to afford 3-oxopropyl groups that eliminate diverse functionalities. The study shows that the reaction is rapid and can liberate phenols and amines near-quantitatively under physiological conditions. The reaction is compatible with living organisms as demonstrated by the release of a resorufin fluorophore and a mexiletine drug in zebrafish embryos implanted with tetrazine-modified beads. The combined benefits of synthetic ease, rapid kinetics, diversity of leaving groups, high release yields, and structural compactness, make 3-isocyanopropyl derivatives attractive chemical caging moieties for uses in chemical biology and drug delivery.

Bioorthogonal dissociative reactions boast diverse potential applications in chemical biology and drug delivery. The reaction of benzonorbornadienes with tetrazines to release amines from carbamate leaving groups was recently introduced as a bioorthogonal bond-cleavage reaction. The present study aimed at investigating the scope of leaving groups that are compatible with benzonorbornadienes. Synthesis of several benzonorbornadienes with different releasable groups is reported, and the reaction of these molecules with tetrazine was found to be rapid and afforded high release yields. The tetrazine-induced release of molecules proceeds in a cascade of steps including inverse-electron demand cycloaddition and cycloreversion reactions that form unstable isoindoles/isobenzofuran intermediates and spontaneously eliminate a leaving group of interest. In the case of oxygen-bridged BNBDs at room temperature, we observed the formation of an unproductive byproduct.

A novel class of bioorthogonal release reactions based on benzonorbornadiene derivatives was developed. These carrier molecules are highly stable at physiological conditions, but react rapidly with 1,2,4,5-tetrazines, and near-quantitatively release cargo molecules such as drugs and optical reporters.

DNA-encoded chemical libraries (DECLs) are pools of DNA-tagged small molecules that enable facile screening and identification of bio-macromolecule binders. The successful development of DECLs has led to their increasingly important role in drug development, and screening hits have entered clinical trials. In this review, we summarize the development and currently active research areas of DECLs with a focus on contributions from groups at academic institutes. We further look at opportunities and future directions of DECL research in medicinal chemistry and chemical biology based on the symbiotic relationship between academia and industry. Challenges associated with the application of DECLs in academic drug discovery are further discussed., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oligonucleotide conjugates of small molecules are widely used in chemical biology and have found increasing interest in the context of DNA-encoded chemical libraries for drug discovery. Attachment of molecules to DNA bound to the solid support is an attractive small-molecule conjugation method that permits the use of organic solvents, rigorous reaction conditions, and simple workup. However, the conjugated structures must be resistant to the harsh DNA deprotection/cleavage conditions and the stabilities of building blocks under various deprotection conditions are mostly unexplored. In the present study, we analyzed the stability of 131 structurally diverse fragments that contain amides and amide-like elements during DNA deprotection protocols. Structural features susceptible to decomposition in DNA deprotection conditions were identified and a protocol that enabled the synthesis of DNA conjugates with labile fragments on solid support was identified.

In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe the synthesis and screening of a DNA-encoded chemical library containing 76230 compounds. In this library, sets of amines and carboxylic acids are directly linked producing encoded compounds with compact structures and drug-like properties. Affinity screening of this library yielded inhibitors of the potential pharmaceutical target tankyrase 1, a poly(ADP-ribose) polymerase. These compounds have drug-like characteristics, and the most potent hit compound (X066/Y469) inhibited tankyrase 1 with an IC50 value of 250 nM.

Parallel affinity screening of a DNA-encoded chemical library against rat, bovine and human serum albumin allowed the identification of small-molecule ligands with distinctive binding specificities to the individual proteins.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5' and 3' ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

An important advantage of pattern-based chemosensor sets is their potential to detect and differentiate a large number of analytes with only few sensors. Here we test this principle at a conceptual limit by analyzing a large set of metal ion analytes covering essentially the entire periodic table, employing fluorescent DNA-like chemosensors on solid support. A tetrameric "oligodeoxyfluoroside" (ODF) library of 6561 members containing metal-binding monomers was screened for strong responders to 57 metal ions in solution. Our results show that a set of 9 chemosensors could successfully discriminate the 57 species, including alkali, alkaline earth, post-transition, transition, and lanthanide metals. As few as 6 ODF chemosensors could detect and differentiate 50 metals at 100 μM; sensitivity for some metals was achieved at midnanomolar ranges. A blind test with 50 metals further confirmed the discriminating power of the ODFs.

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

Heavy metal contamination of water can be toxic to humans and wildlife; thus the development of methods to detect this contamination is of high importance. Here we describe the design and application of DNA-based fluorescent chemosensors on microbeads to differentiate eight toxic metal ions in water. We developed and synthesized four fluorescent 2'-deoxyribosides of metal-binding ligands. A tetramer-length oligodeoxy-fluoroside (ODF) library of 6561 members was constructed and screened for sequences responsive to metal ions, of which seven sequences were selected. Statistical analysis of the response patterns showed successful differentiation of the analytes at concentrations as low as 100 nM. Sensors were able to classify water samples from 13 varied sites and quantify metal contamination in unknown specimens. The results demonstrate the practical potential of bead-based ODF chemosensors to analyze heavy metal contamination in water samples by a simple and inexpensive optical method., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

Templated fluorescence activation has recently emerged as a promising molecular approach to detect and differentiate nucleic acid sequences in vitro and in cells. Here, we describe the application of a reductive quencher release strategy to the taxonomic analysis of Gram-negative bacteria by targeting a single nucleotide difference in their 16S rRNA in a two-color assay. For this purpose, it was necessary to develop a release linker containing a quencher suitable for red and near-infrared fluorophores, and to improve methods for the delivery of probes into cells. A cyanine-dye labeled oligonucleotide probe containing the new quencher-release linker showed unprecedentedly low background signal and high fluorescence turn-on ratios. The combination of a fluorescein-containing and a near-IR emitting probe discriminated E. coli from S. enterica despite nearly identical ribosomal target sequences. Two-color analysis by microscopy and the first successful discrimination of bacteria by two-color flow cytometry with templated reactive probes are described.

We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR (STAR = Staudinger-triggered α-azidoether release) probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe successively binds adjacent to two mono-triphenylphosphine-(TPP)-DNAs or one dual-TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

RNA-templated fluorescence activation is a nucleic acid detection strategy that offers the possibility of direct visual detection of genetic information in living cells. Here we describe a new reaction strategy for fluorescence activation in which a phosphine on one DNA probe reduces an azide group in a linker on a second probe, resulting in linker cleavage and release of a fluorescence quenching group. These "Q-STAR" probes are shown to yield a strong fluorescence turn-on signal in approximately 20 min, with very low background and substantial amplification by turnover on the template. A green/red pair of such probes allowed the discrimination of two bacterial species by a single nucleotide difference in their 16S rRNA. The beneficial properties of the reductive quencher release design make these probes promising candidates for widespread application in the detection of nucleic acids in vitro and in cells.

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.

A nucleic acid detection scheme that employs DNA-mediated delivery of an organomercury activator to unmask a fluorophore is described. The approach relies on adjacent hybridization of two oligonucleotide conjugates containing organomercury and caged rhodamine functionalities. Postsynthetic conjugation of amino-modified DNAs enabled efficient preparation of these probes. Complementary DNA templates yielded fluorescence signals arising from metal-assisted rhodamine uncaging.

Substitution of natural nucleobases in PNA oligomers with ligands is a strategy for directing metal ion incorporation to specific locations within a PNA duplex. In this study, we have synthesized PNA oligomers that contain up to three adjacent bipyridine ligands and examined the interaction with Ni2+ and Cu2+ of these oligomers and of duplexes formed from them. Variable-temperature UV spectroscopy showed that duplexes containing one terminal pair of bipyridine ligands are more stable upon metal binding than their nonmodified counterparts. While binding of one metal ion to duplexes that contain two adjacent bipyridine pairs makes the duplexes more stable, additional metal ions lower the duplex stability, with electrostatic repulsions being, most likely, an important contributor to the destabilization. UV titrations showed that the presence of several bipyridine ligands in close proximity of each other in PNA oligomers exerts a chelate effect. A supramolecular chelate effect occurs when several bipyridines are brought next to each other by hybridization of PNA duplexes. EPR spectroscopy studies indicate that even when two Cu2+ ions coordinate to a PNA duplex in which two bipyridine pairs are next to each other, the two metal-ligand complexes that form in the duplex are far enough from each other that the dipolar coupling is very weak. EXAFS and XANES show that the Ni2+-bipyridine bond lengths are typical for [Ni(bipy)2]2+ and [Ni(bipy)3]2+ complexes.