Ruijie Deng

Toehold‐initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA-mediated regulatory circuits and their disease associations. A toehold-initiated strand-displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold-initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.

Isothermal Amplification for MicroRNA Detection: From the Test Tube to the Cell

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as pivotal post-transcriptional regulators of gene expression, thus involving in many fundamental cellular processes such as cell proliferation, migration, and canceration. The detection of miRNAs has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Particularly, miRNAs in peripheral blood have recently been recognized as important biomarkers potential for liquid biopsy. Furthermore, as miRNAs are expressed heterogeneously in different cells, investigations into single-cell miRNA expression will be of great value for resolving miRNA-mediated regulatory circuits and the complexity and heterogeneity of miRNA-related diseases. Thus, the development of miRNA detection methods, especially for complex clinic samples and single cells is in great demand. In this Account, we will present recent progress in the design and application of isothermal amplification enabling miRNA detection transition from the test tube to the clinical sample and single cell, which will significantly advance our knowledge of miRNA functions and disease associations, as well as its translation in clinical diagnostics. miRNAs present a huge challenge in detection because of their extremely short length (∼22 nucleotides) and sequence homology (even with only single-nucleotide variation). The conventional golden method for nucleic acid detection, quantitative PCR (qPCR), is not amenable to directly detecting short RNAs and hardly enables distinguishing between miRNA family members with very similar sequences. Alternatively, isothermal amplification has emerged as a powerful method for quantification of nucleic acids and attracts broad interest for utilization in developing miRNA assays. Compared to PCR, isothermal amplification can be performed without precise control of temperature cycling and is well fit for detecting short RNA or DNA. We and other groups are seeking methods based on isothermal amplification for detecting miRNA with high specificity (single-nucleotide resolution) and sensitivity (detection limit reaching femtomolar or even attomolar level). These methods have recently been demonstrated to quantify miRNA in clinical samples (tissues, serum, and plasma). Remarkably, attributed to the mild reaction conditions, isothermal amplification can be performed inside cells, which has recently enabled miRNA detection in single cells. The localized in situ amplification even enables imaging of miRNA at the single-molecule level. The single-cell miRNA profiling data clearly shows that genetically identical cells exhibit significant cell-to-cell variation in miRNA expression. The leap of miRNA detection achievements will significantly contribute to its full clinical adoption and translation and give us new insights into miRNA cellular functions and disease associations.

Multiresponsive Rolling Circle Amplification for DNA Logic Gates Mediated by Endonuclease

Rolling circle amplification (RCA), an efficient isothermal amplification method allowing the polymerase-mediated generation of long single-stranded DNA molecules made of tandem repeats, has been widely used in biomedical and nanotechnology fields due to structural and compositional versatility of its components. In this work, we confer multiresponsiveness to RCA reactions by designing dumbbell-shaped DNA templates and hairpin probes containing different endonuclease cleavage sites. Endonucleases trigger the release of RCA primers or the cleavage of DNA templates, which controls subsequent RCA reactions. A set of one-input and two-input DNA logic gates, which use endonucleases or hairpin probes as inputs, including YES, NOT, AND, OR, NOR, and INHIBIT, are constructed on the basis of our proposed multiresponsive RCA reactions. We demonstrate flexibility and scalability of these logic gates by integrating them to fabricate more complex three-input logic circuits (AND-OR and NOR-AND circuits). Moreover, our strategy is used to construct an assay system for endonuclease activity. Our proposed method might be applicable in the multichannel detection of endonucleases, nucleic acids, and other biomolecules.

Direct Visualization of Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation Assay

The accumulation of mitochondrial DNA (mtDNA) mutations in cells is strongly related to aging-associated diseases. Imaging of single-nucleotide variation (SNV) in mtDNA is crucial for understanding the heteroplasmy of mtDNAs that harbor pathogenic changes. Herein, we designed a CRISPR/Cas9-mediated proximity ligation assay (CasPLA) for direct visualization of the ND4 and ND5 genes in the mtDNAs of single cells. Taking advantage of the high specificity of CRISPR/Cas9, CasPLA can be used to image SNV in the ND4 gene at single-molecule resolution. Using CasPLA, we observed a mtDNA-transferring process between different cells through a tunneling nanotube, which may account for the spreading of mtDNA heteroplasmy. Moreover, we demonstrated that CasPLA strategy can be applied for imaging of single copy genomic loci ( KRAS gene) in the nuclear genome. Our results establish CasPLA as a tool to study SNV in situ in single cells for basic research and genetic diagnosis.

Amplified Tandem Spinach-Based Aptamer Transcription Enables Low Background miRNA Detection

MicroRNAs (miRNAs) play key roles in regulating gene expression and cell functions, which are recognized as potential biomarkers for many human diseases. Sensitive, specific, and reliable detection of miRNA is highly demanded for clinical diagnosis and therapy. Herein, we describe a label-free and low-background fluorescent assay, termed amplified tandem Spinach-based aptamer transcription assay (AmptSpi assay) for highly sensitive miRNA detection by polymeric rolling circle amplicon mediated multiple transcription. Target miRNA is recognized by padlock probe to form polymeric rolling circle amplicon. Then the following transcription process rapidly produces large amounts of repeats of RNA Spinach aptamers, lightened up by the addition of fluorescent dye DFHBI for miRNA quantitative analysis, achieving label-free and nearly zero-background. Besides, the assay could also confer high selectivity to distinguish miRNA among the miRNA family members with 1- or 2-nucleotide (nt) difference. This method was capable of completing detection in human serum sample or cell extracts in hours, indicating great potential in the early diagnosis of diseases.

SpliceRCA: in Situ Single-Cell Analysis of mRNA Splicing Variants

Immune cell heterogeneity due to the differential expression of RNA splicing variants still remains unexplored. This is mainly because single-cell imaging technologies of splicing variants with precise sequence or base resolution are now not readily available. Herein, we design a splice-junction anchored padlock-probe-mediated rolling circle amplification assay (SpliceRCA) for single-cell imaging of splice isoforms of essential regulatory immune gene (CD45) upon T-cell activation. Two recognition regions in the padlock probe can target the splice-junction sequence, resulting in a close proximity for triggering in situ one-target-one-amplicon amplification. With the read length of ∼30 nucleotides, this method allows discrimination of isoforms with single-base precision and quantification of isoforms with single-molecule resolution. We applied SpliceRCA to single-cell image splice variants of essential regulatory immune gene (CD45) upon T-cell activation. It is found that CD45RO isoform presents a distal nuclear spatial distribution and is coregulated with CD45RB upon activation. Our strategy provides a single-cell analysis platform to investigate the mechanism of complex immune responses and may further guide immunotherapy.