Universal Exponential Amplification Confers Multilocus Detection of Mutation-Prone Virus.

The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread rapidly around the world. Accurate and scalable diagnostics are essential for immediate intervention and control of viral transmission. Currently reported diagnostics are rapid and sensitive, yet most are limited by their principle of single-locus identification and suffer from false-negative results because of the mutation-prone nature of RNA viruses. Here, we propose a multilocus detection method for SARS-CoV-2 based on a modified loop-mediated isothermal amplification with a pair of universal primers. The sequence-specific probes are designed to recognize the sequence of nucleocapsid protein (N) and the open reading frame 1ab (Orf1ab) gene from the SARS-CoV-2 genome. In the presence of a target locus, separated probes are ligated to be an intact template, the bipartite ends of which are repetitive sequences for the sequential binding of universal primers to initiate strand displacement. A kind of flap structure-dependent endonuclease is involved in cleaving multicolor TaqMan probes during multiplex amplification, realizing a real-time and multiplex analysis. We evaluated the quantitative performance of the developed method with spiked samples using synthetic target RNA, resulting in a limit of detection as low as 250 aM. Furthermore, the feasibility of multilocus detection was validated using various mutation-prone genes, demonstrating a significant potential for accurate analysis of SARS-CoV-2 and holding great promise for the clinical diagnosis of other infectious diseases.

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

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.

RNA-Primed Amplification for Noise-Suppressed Visualization of Single-Cell Splice Variants.

Splice variants visualization is pivotal for a deeper understanding of cell growth and development. However, it remains technically challenging due to short lengths, similar sequences, and low abundance. The existing single-cell imaging strategies suffer from nonspecific amplification that causes considerable noise during visualization of the splice variants. Herein we develop a new RNA-primed amplification strategy for noise-suppressed visualization of single-cell splice variants. Block probes were designed to specifically identify the conjugated region of exons in mRNA, which was then digested by endonuclease and provided a hydroxyl group at the 3' terminal. The RNA target can act as primer to trigger rolling circle amplification, achieving visualization of splice variants with noise suppressed to nearly zero. We further explored the expression and distribution of BRCA1 splice variants in three breast cell lines, revealing cell-type specific mapping of this cancer suppressor gene.

A bifunctional chemical signature enabling RNA 4-thiouridine enrichment sequencing with single-base resolution.

Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules via click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.