Imaging metabolite dynamics in living cells using a Spinach-based riboswitch.

Riboswitches are natural ligand-sensing RNAs typically that are found in the 5' UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5'-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches.

Reproducibility of a commercially available subgingival plaque sampling strategy and analysis strategy with oligonucleotide probes.

OBJECTIVES: The aim was to evaluate the intra-test agreement of pooled samples from the deepest periodontal pocket of each quadrant with a commercially available test kit based on hybridization of 16S rRNA. MATERIAL AND METHODS: Plaque samples of 50 patients with generalized severe chronic periodontitis before therapy were pooled in two separate vials in order to detect and compare counts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Cohen's κ and interclass correlation coefficients were calculated to judge intra-test agreement. RESULTS: Cohen's κ for detection and counts of Tannerella forsythia and Treponema denticola showed a perfect agreement. Porphyromonas ginigivalis was identified in both tests with a substantial agreement, whereas detection of Aggregatibacter actinomycetemcomitans varied in eight patients resulting in a good agreement. Possible confounding factors could not be identified statistically. CONCLUSION: Test results of the commercial 16S rRNA test are perfectly reproducible regarding detection of red complex pathogens. Intra-test agreement concerning detection of Aggregatibacter actinomycetemcomitans was less favorable. CLINICAL RELEVANCE: Detection of certain periodontal pathogens may alter the treatment and lead to prescription of antibiotics parallel to mechanical debridement. It is quite important not to use antibiotics excessively. Thus, the basis for decision-making in favor of antibiotics should be solid.

Proximal disruptor aided ligation (ProDAL) of kilobase-long RNAs.

RNA with site-specific modification is a useful tool for RNA biology studies. However, generating kilobase (kb) -long RNA with internal modification at a site distant from RNA termini remains challenging. Here we report an enhanced splint ligation technique, proximal disruptor aided ligation (ProDAL), which allows adequate efficiency toward this purpose. The key to our approach is using multiple DNA oligonucleotides, 'proximal disruptors', to target the RNA substrate sequence next to the ligation site. The binding of disruptors helps to free the ligation site from intramolecular RNA basepairing, and consequently promotes more efficient formation of the pre-ligation complex and a higher overall ligation yield. We used naturally occurring 1.0 kb renilla and 1.9 kb firefly luciferase mRNA sequences to test the efficacy of our approach. ProDAL yielded 9-14% efficiency for the ligation between two RNA substrates, both of which were between 414 and 1313 nucleotides (nt) long. ProDAL also allowed similarly high efficiency for generating kb-long RNA with site-specific internal modification by a simple three-part ligation between two long RNA substrates and a modification-carrying RNA oligonucleotide. In comparison, classical splint ligation yielded a significantly lower efficiency of 0-2% in all cases. We expect that ProDAL will benefit studies involving kb-long RNAs, including translation, long non-coding RNAs, RNA splicing and modification, and large ribonucleoprotein complexes.

2'-Bispyrene-modified 2'-O-methyl RNA probes as useful tools for the detection of RNA: synthesis, fluorescent properties, and duplex stability.

The synthesis and properties two series of new 2'-O-methyl RNA probes, each containing a single insertion of a 2'-bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21-fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5'-side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3'-side are important: CC, CG, and UC dinucleotide units on the 3'-side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2'-bispyrene-labeled 2'-O-methyl RNA probes might be useful tools for detection of RNAs.

Northern blotting of endogenous full-length human-specific LINE-1 RNA.

LINE-1 belongs to a family of DNA elements that move to new locations in the genome in a process called "retrotransposition." This is achieved by a copy-and-paste mechanism with the aid of an RNA intermediate. The full-length LINE-1 is responsible for most retrotransposition activity in the human genome. Detecting the active LINE-1 RNA at the endogenous level is challenging due to its small percentage among inactive copies and its different forms of transcripts. Here, we describe a method of designing RNA probes to detect active LINE-1 by northern blotting and use optimized conditions and tools to make the detection practical. This method uses a classical long RNA probe and provides an alternative way to detect LINE-1 RNA using multiple short RNA probes.

RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs.

Recent Advancements in the Application of Circulating Tumor DNA as Biomarkers for Early Detection of Cancers.

Early detection of cancer is vital for increasing patient survivability chances. The three major techniques used to diagnose cancers are instrumental examination, tissue biopsy, and tumor biomarker detection. Circulating tumor DNA (ctDNA) has gained much attention in recent years due to advantages over traditional technology, such as high sensitivity, high specificity, and noninvasive nature. Through the mechanism of apoptosis, necrosis, and circulating exosome release in tumor cells, ctDNA can spread throughout the circulatory system and carry modifications such as methylations, mutations, gene rearrangements, and microsatellite instability. Traditional gene-detection technology struggles to achieve real-time, low-cost, and portable ctDNA measurement, whereas electrochemical biosensors offer low cost, high specificity alongside sensitivity, and portability for the detection of ctDNA. Therefore, this review focuses on describing the recent advancements in ctDNA biomarkers for various cancer types and biosensor developments for real-time, noninvasive, and rapid ctDNA detection. Further in the review, ctDNA sensors are also discussed in regards to their selections of probes for receptors based on the electrode surface recognition elements.

Targeted 16S rRNA Gene Capture by Hybridization and Bioinformatic Analysis.

Next-generation sequencing technologies have impressively unlocked capacities to depict the complexity of microbial communities. Microbial community structure is for now routinely monitored by sequencing of 16S rRNA gene, a phylogenetic marker almost conserved among bacteria and archaea. Nevertheless, amplicon sequencing, the most popular used approach, suffers from several biases impacting the picture of microbial communities. Here, we describe an innovative method based on gene capture by hybridization for the targeted enrichment of 16S rDNA biomarker from metagenomic samples. Coupled to near full-length 16S rDNA reconstruction, this approach enables an exhaustive and accurate description of microbial communities by enhancing taxonomic and phylogenetic resolutions. Furthermore, access of captured 16S flanking regions opens link between structure and function in microbial communities.

Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae.

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose-inducing promoter (GAL1) in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL1 promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.

Studying Hemichordata WBR Using Ptychodera flava.

Hemichordates are benthic marine invertebrates closely related to chordates. Several species, including Ptychodera flava in the phylum Hemichordates, can undergo whole body regeneration from a small fragment. P. flava is widely distributed in the warm Indo-Pacific region and is easily collected in the lower tidal zone of a shallow beach with a coral reef. Here, we describe the methods for animal collection and preparation of regenerating tissues. The prepared tissues can be used for various molecular and/or histological experiments. We also demonstrate how to examine gene expression patterns in the tissues using whole mount in situ hybridization.

Naturally occurring three-way junctions can be repurposed as genetically encoded RNA-based sensors.

Small molecules can be imaged in living cells using biosensors composed of RNA. However, RNA-based devices are difficult to design. Here, we describe a versatile platform for designing RNA-based fluorescent small-molecule sensors using naturally occurring highly stable three-way junction RNAs. We show that ligand-binding aptamers and fluorogenic aptamers can be inserted into three-way junctions and connected in a way that enables the three-way junction to function as a small-molecule-regulated fluorescent sensor in vitro and in cells. The sensors are designed so that the interhelical stabilizing interactions in the three-way junction are only induced upon ligand binding. We use these RNA-based devices to measure the dynamics of S-adenosylmethionine levels in mammalian cells in real time. We show that this strategy is compatible with diverse metabolite-binding RNA aptamers, fluorogenic aptamers, and three-way junctions. Overall, these data demonstrate a versatile method for readily generating RNA devices that function in living cells.

In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH).

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins' distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity.

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.

Extracellular vesicle microRNA quantification from plasma using an integrated microfluidic device.

Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (<10% uncertainty) of both free-floating miRNAs and EV-miRNAs in plasma with 1 pM detection sensitivity. The assay time is only 30 minutes as opposed to 13 h and requires only ~20 µL of sample as oppose to 1 mL for conventional RT-qPCR techniques. The platform integrates a surface acoustic wave (SAW) EV lysing microfluidic chip with a concentration and sensing microfluidic chip incorporating an electrokinetic membrane sensor that is based on non-equilibrium ionic currents. Unlike conventional RT-qPCR methods, this technology does not require EV extraction, RNA purification, reverse transcription, or amplification. This platform can be easily extended for other RNA and DNA targets of interest, thus providing a viable screening tool for early disease diagnosis, prognosis, and monitoring of therapeutic response.

Detection of Low-Abundance Metabolites in Live Cells Using an RNA Integrator.

Genetically encoded biosensors are useful tools for detecting the presence and levels of diverse biomolecules in living cells. However, low-abundance targets are difficult to detect because they are often unable to bind and activate enough biosensors to detect using standard microscopic imaging approaches. Here we describe a type of RNA-based biosensor, an RNA integrator, which enables detection of low-abundance targets in vitro and in living cells. The RNA integrator is an RNA sequence comprising a ribozyme and an unfolded form of the fluorogenic aptamer Broccoli. Upon binding its target, the ribozyme undergoes cleavage and releases Broccoli, which subsequently folds and becomes fluorescent. Importantly, each target molecule can bind and induce cleavage of multiple copies of the integrator sensor, resulting in an amplified signal. We show that this approach can be generalized to numerous different ribozyme types for the detection of various small molecules.

Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells.

Genetically encoded small-molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small-molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small-molecule analyte. Here, we describe how to design and optimize these sensors by adjusting critical sequence elements, guided by structural insights from the Spinach aptamer. We provide a stepwise procedure to characterize sensors in vitro and to express sensors in bacteria for live-cell imaging of metabolites. Spinach riboswitch sensors offer a simple method for fluorescence measurement of a wide range of metabolites for which riboswitches exist, including nucleotides and their derivatives, amino acids, cofactors, cations, and anions.

In situ hybridization to detect and localize signature cytokines of T-helper (Th) 1 and Th2 immune responses in chicken tissues.

The avian immune system has been shown to possess a repertoire of cytokines directing T-helper (Th) 1 and Th2 types of immune responses similar to that in mammals. The objective of this study was to establish in situ hybridization (ISH) for the localization of mRNA of selected signal cytokines, chicken interferon-γ (ChIFN-γ), chicken interleukin (ChIL)-4 and ChIL-13 in fixed tissues. RNA probes were generated to hybridize to 488, 318, and 417bp of the respective target mRNA. Probe concentrations ranging from 100ng/ml to 400ng/ml were shown to be suitable to label cells that expressed these cytokines. The specificity of every probe was verified using the respective sense probe. ChIFN-γ, ChIL-4 and ChIL-13 positive cells were observed in the lymphocytic infiltrations of liver and in the periarteriolar lymphatic sheaths of spleen collected from specific-pathogen-free chickens. ISH of these cytokines in a severely inflamed liver due to infiltration with the parasite Histomonas meleagridis revealed the expression of both ChIFN-γ and ChIL-13 mRNA in the mononuclear infiltrates. In conclusion, ChIFN-γ, ChIL-4 and ChIL-13 mRNA were efficiently localized by ISH, which supplies a valid technique to characterize immune responses in fixed tissues.

Levels of Candidate Periodontal Pathogens in Subgingival Biofilm.

In recent years, several new periodontal taxa have been associated with the etiology of periodontitis. A recent systematic review provides further support for the pathogenic role of 17 species/phylotypes. Thus, the aim of this study was to assess the prevalence and levels of these species in subjects with generalized chronic periodontitis (GChP; n = 30), generalized aggressive periodontitis (GAgP; n = 30), and periodontal health (PH; n = 30). All subjects underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analyzed for their content of 20 bacterial species/phylotypes through the RNA-oligonucleotide quantification technique. Subjects from the GChP and GAgP groups presented the highest mean values for all clinical parameters in comparison with the PH group (P < 0.05). Subjects with GChP and GAgP showed significantly higher mean levels of Bacteroidetes sp. human oral taxon (HOT) 274, Fretibacterium sp. HOT 360, and TM7 sp. HOT 356 phylotypes, as well as higher mean levels of Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas gingivalis, Tannerella forsythia, and Selenomonas sputigena species than PH subjects (P < 0.05). GAgP subjects presented higher mean levels of TM7 sp. HOT 356 and F. alocis than GChP subjects (P < 0.05). A significantly higher mean prevalence of Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, and Fretibacterium sp. HOT 362 was found in subjects with GChP and GAgP than in PH subjects. Mean levels of P. gingivalis (r = 0.68), T. forsythia (r = 0.62), F. alocis (r = 0.51, P = 0.001), and Fretibacterium sp. HOT 360 (r = 0.41) were correlated with pocket depth (P < 0.001). In conclusion, Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, Fretibacterium sp. HOT 362, and TM7 sp. HOT 356 phylotypes, in addition to F. alocis, F. fastidiosum, and S. sputigena, seem to be associated with periodontitis, and their role in periodontal pathogenesis should be further investigated.

Targeted Gene Capture by Hybridization to Illuminate Ecosystem Functioning.

Microbial communities are extremely abundant and diverse on earth surface and play key role in the ecosystem functioning. Thus, although next-generation sequencing (NGS) technologies have greatly improved knowledge on microbial diversity, it is necessary to reduce the biological complexity to better understand the microorganism functions. To achieve this goal, we describe a promising approach, based on the solution hybrid selection (SHS) method for the selective enrichment in a target-specific biomarker from metagenomic and metatranscriptomic samples. The success of this method strongly depends on the determination of sensitive, specific, and explorative probes to assess the complete targeted gene repertoire. Indeed, in this method, RNA probes were used to capture large DNA or RNA fragments harboring biomarkers of interest that potentially allow to link structure and function of communities of interest.

In Situ Hybridization to Identify Gut Stem Cells.

In recent years, considerable effort has been directed toward identifying the repertoire of genes specifically expressed in adult stem cells. In this unit, we describe an in situ hybridization protocol adapted for the analysis of gene expression in the intestinal mucosa. This methodology allows researchers to quickly visualize the expression profile of putative stem cell markers with a high degree of sensitivity and resolution.