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ChIP followed by next-generation sequencing (ChIP-Seq) is a key technique for mapping the distribution of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge to define a quantitative scale for ChIP-Seq data, and as such, several approaches making use of exogenous additives, or "spike-ins," have recently been developed. Herein, we report on the development of a quantitative, physical model defining ChIP-Seq. The quantitative scale on which ChIP-Seq results should be compared emerges from the model. To test the model and demonstrate the quantitative scale, we examine the impacts of an EZH2 inhibitor through the lens of ChIP-Seq. We report a significant increase in immunoprecipitation of presumed off-target histone PTMs after inhibitor treatment, a trend predicted by the model but contrary to spike-in–based indications. Our work also identifies a sensitivity issue in spike-in normalization that has not been considered in the literature, placing limitations on its utility and trustworthiness. We call our new approach the sans-spike-in method for quantitative ChIP-sequencing (siQ-ChIP). A number of changes in community practice of ChIP-Seq, data reporting, and analysis are motivated by this work.

We synthesized two dTTP analogues for copper-free “click” chemistry-coupling in the active sites of DNA polymerases. We found that in the presence of both analogues, human immunodeficiency virus (HIV) reverse transcriptase (RT) activity was suppressed by up to 93%. This inhibitory effect was not recovered by an excess amount of primer–template unlike that for a conventional HIV RT inhibitor, azidothymidine. This finding may become the basis for the development of efficient in vivo inhibitors of HIV RT and other DNA polymerases.

Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.

International audience; Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases. Since then, the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass-spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification.

Significance DNA methylation and histone posttranslational modifications are key epigenetic marks that contribute to the fine-tuned regulation of gene expression and other chromatin-templated biological processes. Here, we build artificial chromatin templates and reveal key chromatin structural features and epigenetic marks that coordinately regulate the binding and enzymatic activity of the DNA methylation regulator UHRF1. Studying activities of epigenetic regulators in the context of defined chromatin templates, particularly for multidomain histone and DNA binding proteins such as UHRF1, is critical for understanding molecular mechanisms of epigenetic crosstalk and mechanics regulating epigenetic signaling, and for determining how epigenetic dysregulation contributes to human disease., Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is a key technique for mapping the distribution and relative abundance of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge regarding the ability to quantitatively plot ChIP-seq data, and as such, approaches making use of exogenous additives, or “spike-ins” have recently been developed. Relying on the fact that the IP step of ChIP-seq is a competitive binding reaction, we present a quantitative framework for ChIP-seq analysis that circumvents the need to modify standard sample preparation pipelines with spike-in reagents. We also introduce a visualization technique that, when paired with our formal developments, produces a much more rich characterization of sequencing data.

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation., Histone methyltransferase MLL4 is a transcriptional regulator. Here the authors identify the PHD6 finger of MLL4 as a selective reader of the epigenetic modification H4K16ac and show that a subset of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions, which depends on MOF activity.

DNA methylation promotes transcription DNA methylation generally represses transcription, but in some instances, it has also been implicated in transcription activation. Harris et al. identified a protein complex in Arabidopsis that is recruited to chromatin by DNA methylation. This complex specifically activated the transcription of genes that are already mildly transcribed but had no effect on transcriptionally silent genes such as transposable elements. The complex thereby counteracts the repression effect caused by transposon insertion in neighboring genes while leaving transposons silent. Thus, by balancing both repressive and activating transcriptional effects, DNA methylation can act to fine-tune gene expression. Science , this issue p. 1182

MORC3 is linked to inflammatory myopathies and cancer, however the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome, and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for the recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA however is negatively regulated by CW, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing newly identified functions of MORC3 and suggest that its modulation may contribute to human disease.

Mapping lysine methyltransferase substrate selectivity reveals gaps in the proteome-wide curation of lysine methylomes., Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments have not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely-used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the “reproducibility crisis,” demonstrating the need for rigorous, platform-appropriate validation.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: they are non-renewable, vary in performance between lots, and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform as well as polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001, eLife digest Cells are able to regulate the activity of their genes in response to different cues. Genetic information is encoded in DNA and one way to regulate gene activity is to modify the DNA by attaching chemical “epigenetic” markers to it. When a cell divides, these epigenetic markers can be inherited by the daughter cells so that they share the same patterns of gene activity as the parent cell. When the DNA of the parent cell is copied prior to cell division, the epigenetic markers are also copied onto the new DNA. Mistakes in this process are linked to a wide range of diseases in humans, such as cancer and neurological disorders. One type of epigenetic marker is known as a methyl tag and it is added to DNA by certain enzymes in a process called DNA methylation. A protein called UHRF1 is required for human cells to inherit patterns of DNA methylation through cell division. This protein binds to newly copied DNA that lacks some methyl tags as well as to another protein associated with DNA called histone H3. UHRF1 modifies histone H3 by attaching a small protein molecule called ubiquitin to it. This helps to recruit a DNA methylation enzyme to place methyl tags on the newly copied DNA. However, it was not clear how the various properties of UHRF1 allow it to control how DNA methylation is inherited. Harrison et al. addressed this question by studying purified proteins and DNA fragments outside of living cells. The results show that UHRF1 binding to DNA and histone H3 work together to bring UHRF1 to the sites on DNA that require methylation. Further experiments revealed that the methylation pattern on newly copied DNA is able to activate the ability of UHRF1 to place ubiquitin on histone H3. The findings of Harrison et al. reveal a new mechanism by which dividing cells control how DNA methylation is inherited by their daughter cells. A future challenge will be to find out how attaching ubiquitin to histone H3 activates DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.17101.002

Microarray-based proteomic platforms have emerged as valuable tools for studying various aspects of protein function, particularly in the field of chromatin biochemistry. Microarray technology itself is largely unrestricted in regard to printable material and platform design, and efficient multidimensional optimization of assay parameters requires fluidity in the design and analysis of custom print layouts. This motivates the need for streamlined software infrastructure that facilitates the combined planning and analysis of custom microarray experiments. To this end, we have developed ArrayNinja as a portable, open source, and interactive application that unifies the planning and visualization of microarray experiments and provides maximum flexibility to end users. Array experiments can be planned, stored to a private database, and merged with the imaged results for a level of data interaction and centralization that is not currently attainable with available microarray informatics tools.

Along with biocompatibility, chemical stability, and simplicity of structural prediction and modification, deoxyribozyme-based molecular sensors have the potential of an improved detection limit due to their ability to catalytically amplify signal. This study contributes to the understanding of the factors responsible for the limit of detection (LOD) of RNA-cleaving deoxyribozyme sensors. A new sensor that detects specific DNA/RNA sequences was designed from deoxyribozyme OA-II [Chiuman, W.; Li, Y. (2006) J. Mol. Biol. 357, 748-754]. The sensor architecture allows for a unique combination of high selectivity, low LOD and the convenience of fluorescent signal monitoring in homogeneous solution. The LOD of the sensor was found to be approximately 1.6 x 10(-10) M after 3 h of incubation. An equation that allows estimation of the lowest theoretical LOD using characteristics of parent deoxyribozymes and their fluorogenic substrates was derived and experimentally verified. According to the equation, "catalytically perfect" enzymes can serve as scaffolds for the design of sensors with the LOD not lower than approximately 2 x 10(-15) M after 3 h of incubation. A new value termed the detection efficiency (DE) is suggested as a time-independent characteristic of a sensor's sensitivity. The expressions for the theoretical LOD and DE can be used to evaluate nucleic acid and protein enzymes for their application as biosensing platforms.

Molecular logic gates made of DNA have attracted significant attention because of biocompatibility, simple design and their ability to analyze and control biological systems.[1] To fuel further development of the field, applications of DNA-based gates to solve significant biological problems are required. Recently we characterized a set of DNA logic gates (YES, NOT, AND, and OR) and demonstrated their connectivity by designing ANDNOT and XOR operations.[1g,h] The gates used hybridization of DNA strands with a molecular beacon (MB) probe to produce a fluorescent output. Here we demonstrate how DNA logic gates can be applied to solve an important biomedical task of analysis of multiple DNA sequences containing a complex set of mutations.

An ideal sensor should be user-friendly and inexpensive. It should report the presence of a specific analyte with high accuracy in a simple and straightforward assay.[1] An example of a near-ideal sensor is the home pregnancy test, in which the exposure of an immunochromatographic strip to a urine sample containing a pregnancy marker, human chorionic gonadotropin, develops a colored test line. The important characteristics of the sensor include visual signal output that can be detected by the naked eye, and a simple protocol that does not require user expertise. Tests of this kind of facile format are yet to be developed for the detection of bacterial species. At the same time, straightforward, inexpensive and yet reliable bacteria-specific tests are urgently needed to expedite point-of-care (POC) disease diagnosis and monitor environmental or food contaminations. Here, we make use of recent advantages in functional DNA technology to design an assay that contributes to the development of an ‘ideal sensor’ against pathogenic bacteria. The assay combines binary deoxyribozyme (Dz) technology[2] and a peroxidase-like deoxyribozyme (PDz)[3] in a cascade shown in Figure 1. The binary Dz probe consists of two DNA strands Dza and Dzb (Figure 1A). Each strand contains a fragment complementary to the specific segment of bacterial RNA (analyte-binding arm) and a fragment complementary to the inhibited peroxidase-like deoxyribozyme (IPDz) substrate (IPDz-binding arms). IPDz is a stem-loop folded DNA with ribonucleotides embedded in the loop region (Figure 1A). A 5’-end terminal fragment of IPDz contains the G-rich sequence of PDz.[3] Exposure of the two strands to a sample containing the target bacterial RNA results in the cascade of events as follows. Dza and Dzb strands hybridize to the bacterial RNA and re-form a catalytic Dz core (Figure 1B). The RNA-cleaving Dz cleaves IPDz and releases the G-rich PDz fragment. PDz catalyzes oxidation of a colorless organic substrate, such as 3,3’-diaminobenzidine (DAB), into a colored product in the presence of hemin and hydrogen peroxide, thus changing the color of the solution (Figure 1C). Figure 1 Principle scheme for deoxyribozyme cascade. A) Deoxyribozyme sensor consists of two strands of a split RNA-cleaving deoxyribozyme, Dza and Dzb, and an inhibited peroxidase-like deoxyribozyme (IPDz). B) Binding to bacterial RNA re-forms the catalytic core ... In this proof-of-concept study, we used the sequence of a human pathogen Mycobacterium tuberculosis (Mtb)[4] 16S rRNA as a practically significant analyte and demonstrated that the deoxyribozyme cascade is capable of differentiating the RNA from two closely related mycobacterial species. The sequence of 16S rRNA is 100% conserved among all members of the slow-growing Mycobacterium Tuberculosis Complex (MTC), which includes Mtb and the vaccine strain M. bovis BCG, among other species.[4] In this study we used RNA purified from M. bovis BCG strain as a representative of MTC, called through the text MTC RNA. M. smegmatis is a non-pathogenic mycobacterium whose 16S rRNA is ~95% identical to that of members of the MTC.[4] We designed two Dz cascade sensors specific to a 50-nt fragment (nts 181-231) of 16S rRNA from either MTC (Figure 2A) or M. smegmatis (Figure S1A). Figure 2 Deoxyribozyme cascade for the detection of MTC 16S rRNA. A) Design of MTC-specific deoxyribozyme cascade. Sequences of M. smegmatis and MTC 16S rRNA differ by several nucleotide substitutions. Insertions are indicated by a nucleotide above the sequence ... Dz sensor reported by Mokany et al.[2b] was adapted for the cleavage of IPDz (Figure S2). The design was uniform and straightforward for the two binary Dz probes specific to different analytes: IPDz-binding arms and the binary catalytic core of Dza and Dzb were the same for the two sensors. Only the analyte-binding arms of Dza and Dzb were designed complementary to their specific analytes (Figures 2A and S1A). The presence of multiple single nucleotide substitutions, insertions and deletions in the fragment of M. smegmatis RNA ensured high specificity of recognition of MTC RNA by the anti-MTC sensor. It was demonstrated earlier that structural adjustments of split probes allow differentiation of analytes that differ by a single nucleotide.[2] Therefore, the Dz cascade is potentially applicable for the analysis of single nucleotide polymorphisms and point mutations, which opens a venue toward simple and inexpensive sensors for diagnosing human genetic disorders and drug-resistant bacteria. The performance of the two sensors was initially optimized using synthetic DNA analytes corresponding to the targeted sequence of bacterial 16S rRNAs (Table 1). The LOD values quantified based on light absorbance at 500 nm using spectrophotometer (Figures S3, S4) were determined to be 0.4 nM and 0.6 nM for MTC- and M. smegmatis-specific sensors, respectively (Figures S3a and S4a). This LOD is at least 20 times lower than that reported for PDz-based sensor without the Dz cascade.[1d,3a-d] Since an instrument-free detection format is preferable for the POC diagnostics, we explored TLC plate-based signal visualization. For this purpose, 3 μL of the final samples were spotted on a TLC plate. The intensity of the spots correlated well with the absorbance values (Figure S3). We next performed the assay using total bacterial RNA isolated from either M. smegmatis or M. bovis BCG. The RNA preparations contained approximately equal amounts of 16S rRNA, as verified by electrophoretic analysis (Figure S5). For both MTC- and M. smegmatis-specific sensors, the absorbance of the samples increased as a function of the RNA amount added (Figure 2C and S6). The LOD was found to be 4.4 ng/μl and 4.8 ng/μl for MTC- and M. smegmatis-specific sensors, respectively. TLC-based visualization allowed reliable detection of as low as 12.5 ng of total bacterial RNA in a 3 μl-spot (Figures 2B and S1B). Taking into account that a cell of slow-growing Mtb contains ~ 4,000 ribosomes and that rRNA comprises 83% of total bacterial RNA,[7] we estimate that 12.5 ng of total RNA corresponds to ~106 CFU. Our experimental correlation determined ~107 CFU required for isolation of 12.5 ng of total RNA. Ten milliliters of a typical sputum sample of an Mtb-infected individual contains 106-107 CFU,[8] which can be detected with the current detection limit. The detection limit can be further improved by employing multiple deoxyribozyme probes targeting all hyper-variable fragments of both 16S and 23S rRNAs. Next, we tested the ability of the sensors to differentiate between the RNA of closely related species. Both MTC and M. smegmatis-specific sensors produced significant color change only in the presence of their specific analytes (Figures 3 and S7). Moreover, the signal triggered by the non-specific RNA was only at the background level, thus demonstrating excellent selectivity of the assay. Figure 3 Selectivity of MTC- and M. smegmatis-specific deoxyribozyme sensors. TLC plate-based visualization of the samples containing no RNA or 100 ng total RNA from either M. smegmatis or BCG strain of M. bovis. Table 1 Sequences of the oligonucleotides used in this study. Sensors based on cascades of Dzs for the detection of nucleic acids have been reported earlier.[5,6] The distinguishing features of the assay proposed here include the following. (i) Excellent selectivity. Binary deoxyribozyme probes were found to have exceptional discrimination ability toward nucleotide substitutions in nucleic acids.[1d,2] This is due to the possibility to design one of the two analyte-binding arms to be selective. In the present design, the analyte-binding arm of Dzb strand was chosen to target a hyper-variable fragment of 16S rRNA (Figure 2A). (ii) Signal can be detected by the naked eye. Most of the deoxyribozyme cascades rely on fluorescence detection.[5] Although more sensitive than visual assays, the fluorescent output requires a fluorometer for signal registration and, therefore, is less preferred for low-cost POC diagnostics. Visual assays based on RNA-cleaving deoxyribozymes have been reported earlier for the detection of metal ions[9] and recently for the detection of a model DNA analyte.[10] Together with the approach presented in this study, such assays may create a basis for a new generation of POC detection technologies. (iii) The assay does not require protein enzymes. A number of previously reported cascades rely on protein enzymes for signal production.[6] However, proteins are significantly less stable than DNA under ambient conditions. Therefore, eliminating protein enzymes from a POC test is preferable for robust performance, reproducible results and economic shipment options. (iv) Simplicity of the assay procedure. Both RNA-cleavage and H2O2 decomposition reactions of the cascade operate in the same buffer, which eliminates the need of a buffer exchange between the reaction stages. The assay only requires incubation of a bacterial RNA with the reagents for 1 h at 50 °C and then for 30 min at room temperature followed by detection of a visual signal using TLC plates. The TLC plates can be scanned by a conventional scanner for quantification if needed. The hands-on time of the entire procedure is less than 10 min. (v) The assay was shown to work with real samples of a practically significant pathogen. To the best of our knowledge, there is no PCR-free assay that combines all the above properties essential for POC diagnostics. Our current study is focused on eliminating the RNA isolation step, which will further simplify the assay.

Molecular Beacon (MB) probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called 'Operating Cooperatively' (OC), which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E. coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution.