Kaixiang Zhang

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.

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.

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.