Unlabeled RNA Research Articles

In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis.

So far, in Bacillus subtilis, only four trans-encoded and 11 cis-encoded sRNAs and their targets have been investigated in detail, the majority of them in our group (rev. in 1, 2). Here, we describe in vitro methods for the analysis of sRNA/mRNA interactions. All these methods have been either elaborated or significantly improved in our group and successfully applied to characterize a number of sRNA/target mRNA systems in Bacillus subtilis for which we provide examples from our own work. The in vitro methods comprise the synthesis and purification of labeled and unlabeled RNA, the analysis of sRNA/mRNA interactions in electrophoretic mobility shift assays (EMSAs) including the calculation of their apparent binding rate constants (kapp) and equilibrium dissociation constants (Kd), the localization of minimal regulatory regions of an sRNA, the determination of the secondary structures of both interacting RNAs and their complex as well as the analysis of RNA chaperones that may promote the sRNA/mRNA interaction.

Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

Presenting technological workflows

Presenting technological workflows

Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling

In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence.

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

Do it yourself! – Initial experiences with self-synthesized CsTFA for RNA-SIP analyses

Cesium trifluoroacetate (CsTFA) is a gradient medium for isopycnic centrifugation in RNA-based Stable Isotope Probing (RNA-SIP), an important means to link the structure and function of microbial communities. We report a protocol to easily synthesize CsTFA from cesium carbonate (Cs2CO3) and trifluoroacetic acid (TFA) and show that self-synthesized CsTFA performs similarly to commercial CsTFA in the separation of isotopically labelled and unlabelled bacterial RNA.

Single Nucleotide Resolution RNA-Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow.

RNA–protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA–protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g., in cell or tissue samples. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide–RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18O4-γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γ-phosphate to the 5′-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.

Practical Aspects of Sample Preparation and Setup of <sup>1</sup>H <em>R<sub>1ρ</sub></em> Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1ρ experiments with both isotopically labeled and unlabeled RNA samples.

Practical Aspects of Sample Preparation and Setup of <sup>1</sup>H <em>R<sub>1ρ</sub></em> Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1ρ experiments with both isotopically labeled and unlabeled RNA samples.

Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification.

The understanding of the functional importance of RNA has increased enormously in the last decades. This has required research on the RNA molecules themselves, with the concomitant need for obtaining purified RNA samples, such as for structural studies by NMR or other methods. The main method to create labeled and unlabeled RNA, T7 in vitro transcription, suffers from sequence-dependent yield and often low homogeneity for short constructs (<100 nt) and requires laborious purification. Additionally, the design of structured RNA fragments mimicking the structure of a larger biological RNA is often not straightforward. Secondary structure simulations can be used to make reliable predictions about the folding of a particular RNA fragment. In this article, we describe how to design an RNA construct of interest from a larger sequence, and we combine several previously published improvements of the in vitro transcription method, such as the use of 2'-methoxy modifications and dimethyl sulfoxide or the use of tandem repeats, to increase yield and purity of in vitro-transcribed RNA. Together with a high-performance liquid chromatography (HPLC) purification procedure using both reversed-phase ion-pairing and ion-exchange HPLC, we provide a robust protocol to obtain highly pure RNA of short to intermediate length in large quantities. The protocol optimizes yield, especially for RNA starting with nucleotides other than G. At the same time, it is simplified, and the required time is reduced. The protocols described here constitute a versatile pipeline for the production of purified RNA samples and are suitable for users with little experience in liquid chromatography. © 2021 The Authors. Basic Protocol 1: RNA construct design Basic Protocol 2: DNA template production and in vitro transcription Alternate Protocol: Tandem transcription and RNase H cleavage Basic Protocol 3: Reversed-phase ion-pairing HPLC purification Basic Protocol 4: Ion-exchange HPLC purification.

Label-Free Detection of miRNA Using Surface-Enhanced Raman Spectroscopy.

miRNA plays a vital role in many biological processes by regulating the expression of target genes and is considered to be a promising disease biomarker. Although surface-enhanced Raman spectroscopy (SERS) has been widely used for the detection of nucleic acid molecules, obtaining a characteristic SERS signal of unlabeled RNA in a nondestructive state still remains a challenge. Herein, titanium ions were used as aggregating agents to induce the aggregation of silver nanoparticles, forming hot spots, and the fingerprint information on miRNAs was successfully obtained. This method was used to determine the RNA sequence of homopolymeric bases and the peak position of each base in the Raman spectrum. The obtained RNA spectrum was normalized with the signal intensity of ribose at 959 cm-1, and the base contents of a series of mature miRNA sequences were quantified. Subsequently, the characteristic SERS signal of the RNA hybridization event was obtained by studying the process of RNA hairpin structure formation, and the role of the precursor mir-21 in regulating the target streptomycin sulfate was successfully analyzed. The changes in the SERS signal intensity at the interaction site were accurately identified. This method has potential applications in biological functions of miRNAs, molecular diagnosis, disease treatment, and targeted drug design.

Towards safer stable isotope probing - effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.

A putative protein-RNA complex regulates posttranscriptional processing of cytochrome P450 aromatase (CYP19A1) in bovine granulosa cells.

Follicle growth and granulosa cell health are dependent on the secretion of estradiol from granulosa cells. Estradiol is synthesized from androgen precursor by cytochrome P450 aromatase (CYP19A1), and in cattle CYP19A1 messenger RNA has a short half-life but a long (3.5 kb) 3'-untranslated region (3'UTR), suggesting that posttranscriptional regulation may be important for control of enzyme activity. We tested this hypothesis by inserting the CYP19A1 3'UTR and fragments thereof into a reporter vector between the end of the luciferase coding region and the polyadenylation signal. The full-length aromatase 3'UTR suppressed luciferase activity to 10% of control levels, and smaller fragments showed that this inhibitory activity lies between +926 and +1134 of the 3'UTR. Protein-RNA cross-linking experiments revealed that these 3'UTR fragments formed an RNA-protein complex of approximately 70 kDa that was present in granulosa cells but not in corpus luteum, lung, liver, kidney, pancreas, or bladder extracts. The RNA-binding activity was specific to the 3'UTR, as shown by competition experiments with unlabeled RNA, and was present only in 3'UTR constructs that inhibited luciferase activity. These data suggest that posttranscriptional regulation is an important component of the control of CYP19A1 expression and involves protein binding to a specific sequence in the 3'UTR.

A semi-supervised machine learning framework for microRNA classification

BackgroundMicroRNAs (miRNAs) are a family of short, non-coding RNAs that have been linked to critical cellular activities, most notably regulation of gene expression. The identification of miRNA is a cross-disciplinary approach that requires both computational identification methods and wet-lab validation experiments, making it a resource-intensive procedure. While numerous machine learning methods have been developed to increase classification accuracy and thus reduce validation costs, most methods use supervised learning and thus require large labeled training data sets, often not feasible for less-sequenced species. On the other hand, there is now an abundance of unlabeled RNA sequence data due to the emergence of high-throughput wet-lab experimental procedures, such as next-generation sequencing.ResultsThis paper explores the application of semi-supervised machine learning for miRNA classification in order to maximize the utility of both labeled and unlabeled data. We here present the novel combination of two semi-supervised approaches: active learning and multi-view co-training. Results across six diverse species show that this multi-stage semi-supervised approach is able to improve classification performance using very small numbers of labeled instances, effectively leveraging the available unlabeled data.ConclusionsThe proposed semi-supervised miRNA classification pipeline holds the potential to identify novel miRNA with high recall and precision while requiring very small numbers of previously known miRNA. Such a method could be highly beneficial when studying miRNA in newly sequenced genomes of niche species with few known examples of miRNA.

Simultaneous Measurement of Transcriptional and Post-transcriptional Parameters by 3′ End RNA-Seq

SummaryCellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis as well as introducing methodological biases and batch effects that hamper direct comparison. Here, we present a simple approach of concurrent sequencing of S. cerevisiae poly(A)+ and poly(A)− RNA 3′ ends to simultaneously estimate total RNA levels, transcription, and decay rates from the same RNA sample. The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouracil, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions in diverse organisms.

Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach.

The knowledge of structure and dynamics is crucial to explain the function of RNAs. While nuclear magnetic resonance (NMR) is well suited to probe these for complex biomolecules, it requires expensive, isotopically labeled samples, and long measurement times. Here we present SELOPE, a new robust, proton‐only NMR method that allows us to obtain site‐specific overview of structure and dynamics in an entire RNA molecule using an unlabeled sample. SELOPE simplifies assignment and allows for cost‐effective screening of the response of nucleic acids to physiological changes (e.g. ion concentration) or screening of drugs in a high throughput fashion. This single technique allows us to probe an unprecedented range of exchange time scales (the whole μs to ms motion range) with increased sensitivity, surpassing all current experiments to detect chemical exchange. For the first time we could describe an RNA excited state using an unlabeled RNA.

Pinpointing Unlabeled RNA Sequences Within a Protein-RNA Complex with Atomic Force Microscopy

Structural information of biomolecules is crucial to understand their working mechanisms and to develop drugs. However, determining biological structures at sub-nanometer resolution often demands tedious sample preparation and vast instrumentational resources. In addition, although super-resolution microscopy techniques have enabled tracking fluorescent molecules at the nanometer scale, the requisite of labeling fluorophores remains a hurdle in studying the innate structure of biological complexes. We have developed a multicolor microscopy that shows chemical identities on top of topography at sub-nanometer resolution based on atomic force microscopy. We record torsional deflection of T-shaped cantilevers to observe single-molecule interaction force on the microsecond timescale, and by modifying the AFM tip with a probe molecule that generates a distinct force-distance profile for each interaction with multiple targets, multicolor imaging is made possible. Here we present label-free imaging of a histone mRNA-protein complex using a specifically-designed probe DNA tip. Histone mRNAs have a conserved stem-loop (SL) structure which forms a ternary complex with stem-loop binding protein and 3’-5’ exoribonuclease. In the crystal structure, SL is sandwiched in between the two protein domains and both of the 5’- and 3’-flanking sequences of the SL are exposed. We have designed a probe DNA that partially hybridizes to each flank sequence for the specific recognition, and the rupture of the transiently formed duplex marks the position of the flank sequence. Superimposing consecutive images of the same complex has shown that the detection signals for each sequence were separately clustered on the complex. This imaging platform can be used to study structures of biological complexes with minimal sample preparation in the physiologically relevant conditions.

Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq).

RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment. The analysis of the RATE-seq data entails normalization of RNAseq libraries to thiolated RNA spike-ins and nonlinear model fitting to estimate the degradation rate constant for each RNA species.

Determination of Resistant Starch Assimilating Bacteria in Fecal Samples of Mice by In vitro RNA-Based Stable Isotope Probing.

The impact of the intestinal microbiota on human health is becoming increasingly appreciated in recent years. In consequence, and fueled by major technological advances, the composition of the intestinal microbiota in health and disease has been intensively studied by high throughput sequencing approaches. Observations linking dysbiosis of the intestinal microbiota with a number of serious medical conditions including chronic inflammatory disorders and allergic diseases suggest that restoration of the composition and activity of the intestinal microbiota may be a treatment option at least for some of these diseases. One possibility to shape the intestinal microbiota is the administration of prebiotic carbohydrates such as resistant starch (RS). In the present study, we aim at establishing RNA-based stable isotope probing (RNA-SIP) to identify bacterial populations that are involved in the assimilation of RS using anaerobic in vitro fermentation of murine fecal material with stable [U13C] isotope-labeled potato starch. Total RNA from these incubations was extracted, processed by gradient ultracentrifugation and fractionated by density. 16S rRNA gene sequences were amplified from reverse transcribed RNA of high and low density fractions suspected to contain labeled and unlabeled RNA, respectively. Phylogenetic analysis of the obtained sequences revealed a distinct subset of the intestinal microbiota involved in starch metabolism. The results suggest Bacteroidetes, in particular genera affiliated with Prevotellaceae, as well as members of the Ruminococcacea family to be primary assimilators of resistant starch due to a significantly higher relative abundance in higher density fractions in RNA samples isolated after 2 h of incubation. Using high performance liquid chromatography coupled to isotope ratio mass spectrometry (HPLC-IRMS) analysis, some stable isotope label was recovered from acetate, propionate and butyrate. Here, we demonstrate the suitability of RNA-SIP to link specific groups of microorganisms with fermentation of a specific substrate. The application of RNA-SIP in future in vivo studies will help to better understand the mechanisms behind functionality of a prebiotic carbohydrate and its impact on an intestinal ecosystem with potential implications for human health.

Use of a redox probe for an electrochemical RNA–ligand binding assay in microliter droplets

In this work, we report an affordable, sensitive, fast and user-friendly electroanalytical method for monitoring the binding between unlabeled RNA and small compounds in microliter-size droplets using a redox-probe and disposable miniaturized screen-printed electrochemical cells.