Single-cell RNA genomics technologies are revolutionizing biomedical science by profiling single cells with unprecedented resolution, providing fundamental insights into the role of different cellular states and intercellular heterogeneity in health and disease. The combination of single-cell RNA sequencing (scRNA-seq) with metabolic RNA labelling approaches now enables time-resolved monitoring of transcriptional responses for thousands of genes in thousands of individual cells in parallel. This facilitates and accelerates direct characterization of the temporal dimension of biological processes, which has been largely missing in current data. In this Primer, we provide an overview of the various metabolic RNA labelling approaches and their combination with currently available scRNA-seq and multi-omics platforms. We summarize the main challenges in the design of such experiments and discuss the various applications of time-resolved scRNA-seq in vitro and in vivo. We outline the computational tools and challenges to the analyses of the temporal dynamics of transcriptional responses at the single-cell level. We discuss the prospect of integrating data obtained by the respective time-resolved scRNA-seq approaches with complementary methods to elucidate gene regulatory networks that underlie molecular mechanisms. Finally, we discuss open questions and challenges in the field and give our thoughts for future development and applications. The combination of single-cell RNA sequencing with metabolic RNA labelling enables a time-resolved view of transcriptional responses in individual cells. In this Primer, Erhard and Saliba et al. discuss metabolic labelling approaches and how to assess the temporal dynamics of transcriptional responses in different conditions. 

https://www.zora.uzh.ch/id/eprint/224580/1/nihms_1613929.pdf

Time-resolved single-cell RNA-seq using metabolic RNA labelling

Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3’-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors. Investigation of spatial organization and relationships of biomolecules in cellular nanoenvironments is necessary to understand essential biological processes, but methodologically challenging. Here, the authors report cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments of DNA modifications around histone post-translational modifications, and explore the nanoenvironments in different cancer cell lines and clinical specimens.

/pdf/cellular-macromolecules-tethered-dna-walking-indexing-to-3vby934150.pdf

Cellular macromolecules-tethered DNA walking indexing to explore nanoenvironments of chromatin modifications

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 β-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

ConspectusNucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson-Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10-2 to 10-6 of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account.Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundance NAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel beads that encapsulate single cells and their genomic DNA after cell lysis. It realizes the analysis of global (accessible and inaccessible) DNA from the same cells. We find that the global levels of both 5hmC and 5hmU in single cells can distinguish different breast cancer cells. Finally, the current limitations of these strategies and the future development directions are also discussed. We hope that this Account can spark new ideas and invite new efforts from different disciplines for single-cell NAM analysis.

Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification.

Quantitative analysis of 5-hydroxymethylcytosine (5hmC) has remarkable clinical significance to early cancer diagnosis; however, it is limited by the requirement in current assays for large amounts of starting material and expensive instruments requring expertise. Herein, we present a highly sensitive fluorescence method, termed hmC-TACN, for global 5hmC quantification from several nanogram inputs based on terminal deoxynucleotide transferase (TdT)-assisted formation of fluorescent copper (Cu) nanotags. In this method, 5hmC is labeled with click tags by T4 phage β-glucosyltransferase (β-GT) and cross-linked with a random DNA primer via click chemistry. TdT initiates the template-free extension along the primer at the modified 5hmC site and then generates a long polythymine (T) tail, which can template the production of strongly emitting Cu nanoparticles (CuNPs). Consequently, an intensely fluorescent tag containing numerous CuNPs can be labeled onto the 5hmC site, providing the sensitive quantification of 5hmC with a limit of detection (LOD) as low as 0.021% of total nucleotides (S/N = 3). With only a 5 ng input (∼1000 cells) of genomic DNA, global 5hmC levels were accurately determined in mouse tissues, human cell lines (including normal and cancer cells of breast, lung, and liver), and urines of a bladder cancer patient and healthy control. Moreover, as few as 100 cells can also be distinguished between normal and cancer cells. The hmC-TACN method has great promise of being cost effective and easily mastered, with low-input clinical utility, and even for the microzone analysis of tumor models.

Highly Sensitive Fluorescence Detection of Global 5-Hydroxymethylcytosine from Nanogram Input with Strongly Emitting Copper Nanotags

Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiated visualization of single-cell 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity-differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi-level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.

Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks.

Dynamic information of intracellular transcripts is essential to understand their functional roles. Routine RNA-sequencing (RNA-seq) methods only measure RNA species at a steady state and do not provide RNA dynamic information. Here, we develop addition-elimination mechanism-activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, nascent transcripts are metabolically labeled with 4-thiouridine (4sU). The total RNA is treated with N2H4·H2O under aqueous conditions. N2H4·H2O is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) based on an addition-elimination chemistry. C* is regarded as cytosine (C) during the DNA extension process. This 4sU-to-C transition marks nascent transcripts, so it enables sequencing analysis of RNA dynamics. We apply our AENT-seq to investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method uses a simple chemical reaction in aqueous solutions and will be rapidly disseminated with extensive applications.

Addition-Elimination Mechanism-Activated Nucleotide Transition Sequencing for RNA Dynamics Profiling

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Pairwise Proximity-Differentiated Visualization of Single-Cell DNA Epigenetic Marks.

Addition-Elimination Mechanism-Activated Nucleotide Transition Sequencing for RNA Dynamics Profiling