Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation.

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3'-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5' ends (including m7G cap) in high yields (70-100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3'-end labelling with 5'-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

Metabolic RNA labeling for probing RNA dynamics in bacteria.

Metabolic labeling of RNAs with noncanonical nucleosides that are chemically active, followed by chemoselective conjugation with imaging probes or enrichment tags, has emerged as a powerful method for studying RNA transcription and degradation in eukaryotes. However, metabolic RNA labeling is not applicable for prokaryotes, in which the complexity and distinctness of gene regulation largely remain to be explored. Here, we report 2'-deoxy-2'-azidoguanosine (AzG) as a noncanonical nucleoside compatible with metabolic labeling of bacterial RNAs. With AzG, we develop AIR-seq (azidonucleoside-incorporated RNA sequencing), which enables genome-wide analysis of transcription upon heat stress in Escherichia coli. Furthermore, AIR-seq coupled with pulse-chase labeling allows for global analysis of bacterial RNA degradation. Finally, we demonstrate that RNAs of mouse gut microbiotas can be metabolically labeled with AzG in living animals. The AzG-enabled metabolic RNA labeling should find broad applications in studying RNA biology in various bacterial species.

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging - analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry - analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging - digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies.

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Nonspecific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published data sets. We show that mispriming can occur with as little as two bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned data set. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published data sets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP, and GRO-seq. We further show how RT mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to completely avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

Probing RNA structure and interaction dynamics at the single molecule level.

RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.

Enzymatic production of single-molecule FISH and RNA capture probes.

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

CIRDES: an efficient genome-wide method for in vivo RNA-RNA interactome analysis.

Complex RNA-RNA interactions underlie fundamental biological processes. However, a large number of RNA-RNA interactions remain unknown. Most existing methods used to map RNA-RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA-RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3' end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA-RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA-RNA interactome analysis.

RNA-based stable isotope probing (RNA-SIP) to unravel intestinal host-microbe interactions.

The RNA-SIP technology, introduced into molecular microbial ecology in 2002, is an elegant technique to link the structure and function of complex microbial communities, i.e. to identify microbial key-players involved in distinct degradation and assimilation processes under in-situ conditions. Due to its dependence of microbial RNA, this technique is particularly suited for environments with high numbers of very active, i.e. significantly RNA-expressing, bacteria. So far, it was mainly used in environmental studies using microbiotas from soil or water habitats. Here we outline and summarize our application of RNA-SIP for the identification of bacteria involved in the degradation and assimilation of prebiotic carbohydrates in intestinal samples of human and animal origin. Following an isotope label from a prebiotic substrate into the RNA of distinct bacterial taxa will help to better understand the functionality of these medically and economically important nutrients in an intestinal environment.

Effects of free patchy ends in ssDNA and dsDNA on gold nanoparticles in a colorimetric gene sensor for Hepatitis C virus RNA.

The presence or absence and nature of the free patchy ends in DNA sequences has a decisive effect on the performance of colorimetric sensors based on the use of gold nanoparticles (Au NPs). The authors have designed two unmodified gene probes (probe 1: a 19-mer; probe 2: an 18-mer). They are complementary to either half of a 37-mer target derived from the conserved region of Hepatitis C Virus (HCV) RNA. Each probe has further been modified with 10-mer poly(A) and thiol-functionalized 10-mer poly(A) at the 5' positions. Nine combinations of probe and HCV RNA were then designed to investigate the effect of free patchy ends on the stability of citrate-modified Au NPs against salt-induced aggregation which lead to color change from red to blue. The aggregation of Au NPs can be monitored by ratiometric spectroscopy at wavelengths of 520 and 700 nm. The differentiation between HCV RNA and control has also been studied by varying the concentration of probe and analyte. The particle size and zeta potentials were determined before and after aggregation. It is demonstrated that the change in surface charge density of the Au NPs governs the critical coagulation concentration of NaCl. The method presented here can be used to quantify HCV RNA in the 370 nM to 3 µM concentration range, and the detection limit is 500 nM. The results obtained with Au NPs that are chemically non-conjugated with the oligonucleotides have been found to be valuable in rationally devising the design rules for rapid and efficient colorimetric sensing of oligonucleotides. Graphical abstract Schematic representation of the nine combinatorial pairs of oligonucleotides that vary in the length of patchy ends and their position to unearth their effect in rapid gold nanoparticle-based colorimetric gene sensing without time-consuming and expensive thiol-conjugation step.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

Development of double labeling in situ hybridization using RNA probes for genome detection of nervous necrosis virus (NNV).

In situ hybridization (ISH) of genomic segments using RNA-RNA hybrid for nervous necrosis virus (NNV) detection has not been reported yet. The objective of this study was to develop RNA-ISH using RNA probes for the detection of NNV in infects SSN-1 cells or sevenband grouper Hyporthodus septemfasciatus. Two viral RNA segments viz., RNA1 and RNA2 were synthesized by in vitro transcription and labeled with fluorescein UTP and dignoxigenin dUTP, respectively. These labeled RNA probes specifically detected NNV in infected SSN-1 cells. We also applied double labeling RNA-ISH with two-color staining of RNA probes. The results showed that these two viral genomic segments were localized in same regions although RNA1 was also expressed separately. These findings suggest that RNA1 overexpression may be important for sufficient assembly of infectious particles. The RNA-ISH showed that both RNA segments were localized in the tectum opticum, torus semicircualris, cerebellum, thalamus, hypothalamus, and medulla of experimentally infected brain tissues. Especially, RNA segments were highly localized around the ventricle, suggesting that ventricle might play a vital role in the spread of NNV. This technique can be useful for understanding the localization of NNV and the relationship between clinical sign and viral expression.

Unspecific binding of cRNA probe to plaques in two mouse models for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the pathological deposition of amyloid-ß (Aß) protein-containing plaques. Microglia and astrocytes are commonly attracted to the plaques by an unknown mechanism that may involve cell adhesion. One cell adhesion family of proteins, the cadherins, are widely expressed in the central nervous system. Therefore, our study was designed to map the expression of cadherins in AD mouse brains. A particular focus was on plaques because diverse mRNA-species were found in plaques and their surrounding area in brains of AD patients. METHODS: In this study, we used in situ hybridization to visualize cadherin expression in brains of two mouse models for AD (APP/PS1 and APP23). RESULTS: A variable number of plaques was detected in transgenic brain sections, depending on the probe used. Our first impression was that the cadherin probes visualized specific mRNA expression in plaques and that endogenous staining was unaffected. However, control experiments revealed unspecific binding with sense probes. Further experiments with variations in probe length, probe sequence, molecular tag and experimental procedure lead us to conclude that cRNA probes bind generally and in an unspecific manner to plaques. CONCLUSIONS: We demonstrate unspecific binding of cRNA probes to plaques in two mouse models for AD. The widespread and general staining of the plaques prevented us from studying endogenous expression of cadherins in transgenic brain by in situ hybridization.

The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease.

Recent studies have established that mutations or deletions in microRNA (miRNA) processing enzymes resulting in a global decrease of miRNA expression are frequent across cancers and can be associated with a poorer prognosis. While very popular in miRNA profiling studies, it remains unclear whether miRNA microarrays are suited or not to accurately detecting global miRNA decreases seen in cancers. In this work, we analyzed the miRNA profiles of samples with global miRNA decreases using Affymetrix miRNA microarrays following the inducible genetic deletion of Dicer1. Surprisingly, up to a third of deregulated miRNAs identified upon Dicer1 depletion were found to be up-regulated following standard robust multichip average (RMA) background correction and quantile normalization, indicative of normalization bias. Our comparisons of five preprocess steps performed at the probe level demonstrated that the use of cyclic loess relying on non-miRNA small RNAs present on the Affymetrix platform significantly improved specificity and sensitivity of detection of decreased miRNAs. These findings were validated in samples from patients with prostate cancer, where conjugation of robust normal-exponential background correction with cyclic loess normalization and array weights correctly identified the greatest number of decreased miRNAs, and the lowest amount of false-positive up-regulated miRNAs. These findings highlight the importance of miRNA microarray normalization for the detection of miRNAs that are truly differentially expressed and suggest that the use of cyclic loess based on non-miRNA small RNAs can help to improve the sensitivity and specificity of miRNA profiling in cancer samples with global miRNA decrease.

A microwell plate-based multiplex immunoassay for simultaneous quantitation of antibodies to infectious viruses.

Antibodies (Abs) to disease-causing viruses in human blood are important indicators of infection status. While ELISA has been widely used to detect these Abs, a multiplex assay system for simultaneous detection of multiple Abs is still a desirable alternative method for a more efficient screening process because of the lack of multiplexing ability in ELISA. However, as all antibodies are based on immunoglobulin and recognized commonly by the same secondary antibody, it is impossible to multiplex the conventional indirect ELISA in a 96-microwell plate-based platform. To overcome this hurdle, we designed an assay consisting of two steps: capturing target Abs by specific antigens on DNA-encoded gold nanoparticles; and quantifying the target Abs by producing RNase H-mediated detection signals based on the DNA and additional RNA probes. With this newly designed method, we could simultaneously analyze three infectious disease-related Abs, anti-HIV Ab, anti-HCV Ab, and anti-HBV Ab, on the microwell-based platform. The assay performance was evaluated by comparison with ELISA. Furthermore, the accuracy and precision of the assay in a practical application was also estimated by determining the amount of target Abs in human serum solutions.

Differential expression of the alternatively spliced forms of prosaposin mRNAs in rat choroid plexus.

Prosaposin has two distinct profiles. One is a precursor form that is processed into saposins thus promoting lysosomal sphingolipid hydrolase function, whereas the other is an intact form that is not processed into saposins but is abundant in certain tissues and secretory fluids, including the cerebrospinal fluid. In rats, alternative splicing in the prosaposin gene generates mRNAs with and without a 9-base insertion (Pro+9 and Pro+0 mRNAs, respectively). Pro+9 mRNA is reported to be preferentially expressed in tissues in which the intact form of prosaposin dominates, whereas Pro+0 mRNA is preferentially expressed in tissues in which the precursor dominates. The expression patterns of Pro+9 and Pro+0 mRNAs in the rat choroid plexus are examined in the present study. The specificities of 36-mer oligonucleotide probes used to detect the 9-base insertion by in situ hybridization were demonstrated by dot-blot hybridization. Next, these probes were used for in situ hybridization, which showed predominant expression of Pro+0 mRNA and weak expression of Pro+9 mRNA in the choroid plexus. These expression patterns were confirmed by reverse transcription plus the polymerase chain reaction with AlwI restriction enzyme treatment. Expression of the intact form of prosaposin in the choroid plexus was assessed by Western blotting and immunohistochemistry. Because the choroid plexus is responsible for the generation of cerebrospinal fluid containing the intact form of prosaposin, the present study raises the possibility that Pro+0 mRNA is related to the intact form in the choroid plexus and that the alternatively spliced forms of mRNAs do not simply correspond to the precursor and intact forms of prosaposin.

Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins.

Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ∼5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.

Dynamic association-dissociation and harboring of endogenous mRNAs in stress granules.

In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)(+) mRNAs in living mammalian cells using a linear antisense 2'-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1-GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2'-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)(+) mRNAs.

Stabilization of ssRNA on graphene oxide surface: an effective way to design highly robust RNA probes.

RNA probes constitute an important class of functional nucleic acids (FNAs). However, because of their notorious vulnerability to enzymatic degradation, extremely careful and special protocols must be followed when dealing with RNA probes. To fully use the large number of RNA FNAs available for bioanalysis and biomedicine, it is important to explore effective methods to protect RNA probes from enzymatic digestion. In this work, we systematically demonstrate that graphene oxide (GO) can effectively protect RNA probes from enzymatic digestion. Based on this finding, we propose an effective way to design robust RNA biosensors by simply mixing RNA probes with GO for analysis of nucleic acids, proteins, and small molecules. The entire assay is sensitive, selective, rapid, and more importantly, does not require any special protocols. The ability to protect ssRNA from enzymatic digestion by GO offers an exciting new way to stabilize ssRNA, which will not only provide new opportunities to utilize the large number of currently available, yet rarely explored, RNA FNAs for bioanalysis but also offer a new solution to protect important ssRNA molecules, such as microRNA and antisense ssRNA, for a great variety of biomedical applications.