Enzymatic Probing Research Articles

The Trouble with Triples: Elucidating the Behavior of Trinucleotide Repeats in Chromatin

Trinucleotide repeats (TNRs) occur throughout the genome and their expansion can affect varied cellular processes such as gene expression, mRNA processing, and protein folding. TNR expansion, facilitated by formation of stable non-canonical secondary structures (e.g. hairpins), has been linked to several neurodegenerative diseases, such as Huntington's Disease, Myotonic Dystrophy (both caused by expansion of CAG•CTG repeats), and Fragile X Mental Retardation (caused by expansion of CGG•CCG repeats). TNR structure and expansion has been studied in vitro using both oligonucleotides and TNRs incorporated into plasmids; however, genomic DNA is packaged into chromatin, and little is know about the effect of packaging on the ability of repetitive DNA to expand. Previous studies have shown that disease-length, expanded CAG•CTG repeats readily incorporate into the basic unit of chromatin packing, the nucleosome core particle (NCP), composed of 146 base pairs of DNA wrapped around a core of eight histone proteins. However, long CGG•CCG repeats exclude NCP formation. Here, we seek to investigate not only the properties associated with shorter repeats (those that have not expanded) incorporated into NCPs, but also the effect of the flanking, disease-associated gene sequence. To assess the global forces regulating interaction between the TNRs and the histone core, we performed competitive nucleosome exchanges to determine the efficiency with which various TNR sequences incorporate into NCPs. We then used enzymatic and chemical probing to determine the effect of histone occupancy on the TNRs, including the formation of possible non-canonical structures. Understanding the intricacies underlying the interaction between unexpanded TNRs and the histone core is important to understanding how TNRs expand in the genome.

CAG/CTG Repeats Alter the Affinity for the Histone Core and the Positioning of DNA in the Nucleosome

Trinucleotide repeats (TNRs) occur throughout the genome, and their expansion has been linked to several neurodegenerative disorders, including Huntington's disease. TNRs have been studied using both oligonucleotides and plasmids; however, less is know about how repetitive DNA responds to genomic packaging. Here, we investigate the behavior of CAG/CTG repeats incorporated into nucleosome core particles, the most basic unit of chromatin packaging. To assess the general interaction between CAG/CTG repeats and the histone core, we determined the efficiency with which various TNR-containing DNA substrates form nucleosomes, revealing that even short CAG/CTG tracts are robust incorporators. However, the presence of the Huntington gene flanking sequence (htt) decreases the rate of incorporation. Enzymatic and chemical probing revealed repositioning of the DNA in the nucleosome as the number of CAG/CTG repeats increased, regardless of the flanking sequence. Notably, the periodicity of the repeat tract remained unchanged as a function of length and is consistently 10.7 bp per helical turn. In contrast, the periodicity of the nonrepetitive flanking sequence varies and is smaller than the repeat tract at ~10.0-10.5 bp per turn. Furthermore, while the CAG/CTG repeats remain as a canonical duplex in the nucleosome, nucleosome formation causes kinking in a secondary repeat tract in the htt gene, comprised of CCG/CGG repeats. This work highlights the innate ability of CAG/CTG repeats to incorporate and to position in nucleosomes and how that behavior is modulated by the htt flanking sequence. In addition, it illuminates the differences in packaging of healthy and diseased length repeat tracts within the genome.

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family

Rapid antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein with subunits encoded by two branches of the ves multigene family. The ves1α and ves1β gene pair encoding VESA1a and 1b, respectively, are transcribed in a monoparalogous manner from a single locus of active ves transcription (LAT), just one of many quasi-palindromic ves loci. To determine whether this organization plays a role in transcriptional regulation, chromatin structure was first assessed. Limited treatment of isolated nuclei with micrococcal nuclease to assay nucleosomal patterning revealed a periodicity of 156–159bp in both bulk chromatin and specific gene coding regions. This pattern also was maintained in the intergenic regions (IGr) of non-transcribed ves genes. In contrast, the LAT IGr adopts a unique pattern, yielding an apparent cluster of five closely-spaced hypersensitive sites flanked by regions of reduced nucleosomal occupancy. ves loci fall into three patterns of overall sensitivity to micrococcal nuclease or DNase I digestion, with only the LAT being consistently very sensitive. Non-transcribed ves genes are inconsistent in their sensitivity to the two enzymatic probes. Non-linear DNA structure in chromatin was investigated to determine whether unique structure arising as a result of the quasi-palindromic nature of the LAT may effect transcriptional control. The in vitro capacity of ves IGr sequences to adopt stable higher-order DNA structure is demonstrated here, but the presence of such structure in vivo was not supported. Based upon these results a working model is proposed for the chromatin structural remodeling responsible for the sequential expression of ves multigene family members from divergently-organized loci.

Highly specific-glucose fluorescence sensing based on boronic anthraquinone derivatives via the GOx enzymatic reaction

The boronic acid anthraquinones, oHAQB and pHAQB, have been designed, and demonstrated to serve as fluorogenic biosensors for glucose. The sensory molecule, oHAQB, has exhibited the specific-glucose sensing via the GOx enzymatic reaction. In this contribution, the fluorescence changes of oHAQB reasonably correspond to the concentration of glucose upon the conversion of boronic acid to hydroxy based sensor by H2O2 generated from glucose along with GOx enzymatic reaction. Our sensing ensemble was then successfully applied to determine the glucose concentration in the range of 0.08–0.42 mM. The limit of detection (LOD) of oHAQB for glucose detection using the GOx enzymatic probe is approximately 0.011 mM.

Pyrite footprinting of RNA

In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals (OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS2) can produce sufficient OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges.

The ribosome modulates the structural dynamics of the conserved GTPase HflX and triggers tight nucleotide binding

The universally conserved GTPase HflX is a putative translation factor whose GTPase activity is stimulated by the 70S ribosome as well as the 50S but not the 30S ribosomal subunit. However, the details and mechanisms governing this interaction are only poorly understood. In an effort to further elucidate the functional mechanism of HflX, we examined its interaction with the 70S ribosome, the two ribosomal subunits (50S and 30S), as well as its ability to interact with guanine nucleotides in the respective ribosomal complexes using a highly purified invitro system. Binding studies reported here demonstrate that HflX not only interacts with 50S and 70S particles, but also with the 30S subunit, independent of the nucleotide-bound state. A detailed pre-steady-state kinetic analysis of HflX interacting with a non-hydrolyzable analog of mant-GTP, coupled with an enzymatic probing assay utilizing limited trypsinolysis, reveal that HflX·GTP exists in a structurally distinct 50S- and 70S-bound form that stabilizes GTP binding up to 70000-fold and that may represent the "GTPase-activated" state. This activation is likely required for efficient GTP-hydrolysis, and may be similar to that observed in elongation factor G. Results reported here address the surprising low affinity of free HflX for GTP and suggest that cellular HflX will mainly exist in the HflX·GTP·ribosome-bound form. A minimal model for the functional cycle of HflX is proposed.

The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation

The 3′ untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3′ UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5′ and 3′ UTRs, we found that the 3′ UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3′-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3′-ends such as the poly(A) or the 3′ UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3′ UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5′ UTR. In conclusion, we have found that the 3′-end terminal sequence can play a role in regulating the translation of CSFV.

Global or local? Predicting secondary structure and accessibility in mRNAs

Determining the structural properties of mRNA is key to understanding vital post-transcriptional processes. As experimental data on mRNA structure are scarce, accurate structure prediction is required to characterize RNA regulatory mechanisms. Although various structure prediction approaches are available, it is often unclear which to choose and how to set their parameters. Furthermore, no standard measure to compare predictions of local structure exists. We assessed the performance of different methods using two types of data: transcriptome-wide enzymatic probing information and a large, curated set of cis-regulatory elements. To compare the approaches, we introduced structure accuracy, a measure that is applicable to both global and local methods. Our results showed that local folding was more accurate than the classic global approach. We investigated how the locality parameters, maximum base pair span and window size, influenced the prediction performance. A span of 150 provided a reasonable balance between maximizing the number of accurately predicted base pairs, while minimizing effects of incorrect long-range predictions. We characterized the error at artificial sequence ends, which we reduced by setting the window size sufficiently greater than the maximum span. Our method, LocalFold, diminished all border effects and produced the most robust performance.

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.

Aptamer enzymatic cleavage protection assay for the gold nanoparticle-based colorimetric sensing of small molecules

A label-free, homogeneous aptamer-based sensor strategy was designed for the facile colorimetric detection of small target molecules. The format relied on the target-induced protection of DNA aptamer from the enzymatic digestion and its transduction into a detectable signal through the length-dependent adsorption of single-stranded DNA onto unmodified gold nanoparticles (AuNPs). The proof-of-principle of the approach was established by employing the anti-tyrosinamide aptamer as a model functional nucleic acid. In the absence of target, the aptamer was cleaved by the phosphodiesterase I enzymatic probe, leading to the release of mononucleotides and short DNA fragments. These governed effective electrostatic stabilization of AuNPs so that the nanoparticles remained dispersed and red-colored upon salt addition. Upon tyrosinamide binding, the enzymatic cleavage was impeded, resulting in the protection of the aptamer structure. As this long DNA molecule was unable to electrostatically stabilize AuNPs, the resulting colloidal solution turned blue after salt addition due to the formation of nanoparticle aggregates. The quantitative determination of the target can be achieved by monitoring the ratio of absorbance at 650 and 520 nm of the gold colloidal solution. A limit of detection of ∼5 μM and a linear range up to 100 μM were obtained. The sensing platform was further applied, through the same experimental protocol, to the adenosine detection by using its DNA aptamer as recognition tool. This strategy could extend the potentialities, in terms of both simplicity and general applicability, of the aptamer-based sensing approaches.

Enzymatic Probing Analysis of an Engineered Riboswitch Reveals Multiple off Conformations

We investigated the gene regulatory mechanism of a previously engineered riboswitch +thiMN15#19 that turns on gene expression in response to thiamine pyrophosphate (TPP). In vitro enzymatic probing was performed to identify the secondary structures of the OFF conformations predicted by Mfold. Interestingly, enzymatic probing data of the riboswitch and its variants indicated that the riboswitch in its OFF state adopts two distinct structures. Moreover, further in vivo experiments suggested that both OFF structures contribute to the riboswitch function. A deeper understanding of how riboswitches function at the molecular level should enhance our ability to design synthetic riboswitches with new or improved characteristics.

Nanoliter droplet array for microRNA detection based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR

In this paper, a nanoliter droplet array based on enzymatic stem-loop probes ligation and SYBR Green real-time PCR for quantification of microRNA was developed. By employing T4 RNA ligase 2 instead of T4 DNA ligase, we designed simplified stem-loop probes to perform microRNA-templated DNA ligation and reduced the non-specific ligation of T4 DNA ligase. SYBR green I dye was employed instead of TaqMan probes in present miniaturized real-time PCR systems. Specifically, we optimized the dosage of SYBR Green I dye in nanoliter droplet and verified the performance of this system by detecting synthetic mir-122 with a 6 logs dynamic range (from 1.5×105 to 1.5×1010 copies). Linear relationship of the standard curve (R2=0.9997) and high PCR amplification efficiency (96.83%) were obtained under the optimized conditions. We detected the expression of mir-122 across five mouse tissues and the result was consistent with that TaqMan microRNA assay. We think this miniaturized real-time PCR platform reduced the detection cost considerably, thus showing the great potential to quantitative biology.

RNA structure probing dash seq

RNA constitutes the central conduit for information storage, conveyance, and manipulation in biological systems (1, 2). RNAs encode their critical information at two levels. First, the primary sequence directs protein synthesis and contains simple cis-acting elements that bind regulatory factors and other RNAs. Second, most RNAs fold to create complex base-paired and higher order structures with intrinsic regulatory functions. Until recently, it has been difficult or impossible to interrogate the structures of most RNAs, especially in complex biological environments. Ongoing advances in nucleotide-resolution RNA structure probing have made possible increasingly rigorous and quantitative analyses (3), and recent large-scale and whole-genome studies have revealed or better defined rules for how RNA structure regulates translation initiation, protein folding, splicing, and access to protein binding sites (4–6). The clear power of next-generation sequencing (NGS) to transform nucleic acid-based analyses (7, 8) has motivated significant efforts (a scientific dash) to meld chemical and enzymatic probing experiments with NGS readouts (termed seq experiments). The melding of RNA structure probing experiments with NGS readout is a potential marriage made in transcriptome heaven. Two papers in PNAS (9, 10) illustrate important progress toward this highly sought goal.

In silico analysis of IRES RNAs of foot-and-mouth disease virus and related picornaviruses

Foot-and-mouth disease virus (FMDV) uses an internal ribosome entry site (IRES), a highly structured segment of its genomic RNA, to hijack the translational apparatus of an infected host. Computational analysis of 162 type II picornavirus IRES RNA sequences yielded secondary structures that included only base pairs supported by comparative or experimental evidence. The deduced helical sections provided the foundation for a hypothetical three-dimensional model of FMDV IRES RNA. The model was further constrained by incorporation of data derived from chemical modification and enzymatic probing of IRES RNAs as well as high-resolution information about IRES RNA-bound proteins.

Sharing and archiving nucleic acid structure mapping data

Nucleic acids are particularly amenable to structural characterization using chemical and enzymatic probes. Each individual structure mapping experiment reveals specific information about the structure and/or dynamics of the nucleic acid. Currently, there is no simple approach for making these data publically available in a standardized format. We therefore developed a standard for reporting the results of single nucleotide resolution nucleic acid structure mapping experiments, or SNRNASMs. We propose a schema for sharing nucleic acid chemical probing data that uses generic public servers for storing, retrieving, and searching the data. We have also developed a consistent nomenclature (ontology) within the Ontology of Biomedical Investigations (OBI), which provides unique identifiers (termed persistent URLs, or PURLs) for classifying the data. Links to standardized data sets shared using our proposed format along with a tutorial and links to templates can be found at http://snrnasm.bio.unc.edu.

Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors

The 5′ leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used ‘standard’ chemical and enzymatic probes and an ‘RNA SHAPE’ analysis to model the structure of the HIV-1 5′ leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5′ leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: Part I – Synthesis protocol

In this study, a new biosynthetic tracer was developed to characterize the virus retention dynamics of membrane systems. This new tracer is a modified bacteriophage obtained by the grafting of enzymatic probes to an MS2 bacteriophage, one of the smallest non-pathogenic bacteria viruses, with an average diameter of about 30 nm. A protocol for the synthesis and purification of this new tracer was developed in this work. The production of this biosynthetic tracer was first qualitatively shown by a chromatographic characterization and an enzymatic test. The average number of probes grafted per phage was then quantified for three batches of tracers made from the same native phage suspension and the same batch of enzymatic probes. This quantification demonstrated the reproducibility of the synthesis protocol developed.

A new biosynthetic tracer for the inline measurement of virus retention in membrane processes: Part II. Biochemical and physicochemical characterizations of the new tracer

In a previous work, a reproducible procedure to produce a new biosynthetic tracer was developed. This new tracer is an MS2 bacteriophage with enzymatic probes grafted on its surface, which can induce enzymatic activity of the tracer. In this paper, the biochemical and physicochemical characteristics of this new tracer are determined. A protocol was developed to determine the specific enzymatic activity kcat TRACER of the tracer, which was found to be 2.93 ± 0.78 × 10 4 min −1 on average. Physicochemical characterizations of this new tracer showed that it is representative of viruses and may thus be used as a virus surrogate to assess the virus retention of membrane systems inline. Notably, the mean diameter and molecular weight of the tracer were found to be respectively 64.1 ± 0.3 nm and 12140 ± 3654 kDa, which are within the size and molecular weight ranges of pathogenic viruses carried by water. The tracer surface was also studied and revealed the considerable porosity of the grafted probe layer, with a mean porosity of 88%, which could explain why the zeta potential of the tracers (−14.34 ± 1.66 mV) was nearly the same as that of the native MS2 phages. Finally, a comparison between filtration of the reference microorganism used for membrane performance assessment (the MS2 phage) and the tracer suspensions showed the same filtration behaviour.

Structural and functional analysis of the Rous Sarcoma virus negative regulator of splicing and demonstration of its activation by the 9G8 SR protein

Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5′-part is considered as an ESE binding SR proteins. Its 3′-part contains a decoy 5′-splice site (ss), which inhibits splicing at the bona fide 5′-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entire RSV NRS. Structural analysis of other avian NRSs and comparison with all sequenced avian NRSs is in favour of a phylogenetic conservation of the NRS 2D structure. By combination of approaches: (i) in vitro and in cellulo splicing assays, (ii) footprinting assays and (iii) purification and analysis of reconstituted RNP complex, we define a small NRS element retaining splicing inhibitory property. We also demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo. Altogether these data bring new insights on how NRS fine tune splicing activity.