Specificity For Single-base Mismatch Research Articles

A programmable quantum dot nanosensor guided by three-way junction skeleton-mediated cascade signal amplification for sensitive detection of circRNAs in breast cancer

Circular RNAs (circRNAs) are endogenous covalent circular molecules that can regulate various biological processes in eukaryotes, and their dysregulation is associated with tumorigenesis. Herein, we demonstrate the construction of a programmable quantum dot (QD) nanosensor guided by three-way junction (TWJ) skeleton-mediated cascade signal amplification for sensitive detection of circRNA in clinical breast tissues. The TWJ probe contains a TWJ primer and a TWJ template. The presence of circRNA can specifically hybridize with TWJ probe to form a stable TWJ skeleton, initiating cascade signal amplification to produce abundant linker probes. The resultant linker probes can bind with Cy5-modified signal probes and biotinylated capture probes to yield the sandwich duplexes. Eventually, the immobilization of sandwich duplexes on the 605QD surface yields the 605QD-dsDNA-Cy5 nanoassembly, resulting in effective fluorescence resonance energy transfer (FRET). This nanosensor is free from exogenous reverse transcription primers and complicated probes design, and it exhibits high amplification efficiency, single-base mismatch specificity, and near-zero background signal. It can detect circRNA with attomolar sensitivity, measure endogenous circRNA at the single-cell level, and discriminate circRNA level in breast cancer tissues from adjacent normal tissues. Moreover, this nanosensor can be expanded to detect other circRNAs (e.g., circHIPK3) by easily changing the corresponding target recognition sequences of TWJ probes, with potential applications in biomedical research and molecular diagnosis.

Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique

This paper presents a novel clustered regularly interspaced short palindromic repeat (CRISPR)-associated HRCA technique (CART). During the entire detection process of CART, the target DNA is first specifically recognized and cleaved by a pair of Cas9/sgRNA complexes; then, the cleaved product is ligated into circular DNA as the template of HRCA, and the circular DNA is efficiently amplified by HRCA. Therefore, CART has the advantages of Cas9/sgRNA (single-base mismatch specificity) and HRCA (isothermal reaction temperature and high sensitivity). This technique has been verified by detecting various human papillomavirus (HPV) genes with numerous subtypes. In summary, this study provides a new and effective method for the detection of nucleic acids.

A rapid bioanalytical tool for detection of sequence-specific circular DNA and mitochondrial DNA point mutations.

Mutations in mitochondrial DNA (mtDNA) have been an essential cause of numerous diseases, making their identification critically important. The majority of mtDNA screening techniques require polymerase chain reaction (PCR) amplification, enzymatic digestion, and denaturation procedures, which are laborious and costly. Herein, we developed a sensitive PCR-free electrokinetic-based sensor combined with a customized bis-peptide nucleic acid (bis-PNA) and gamma-PNA (γ-PNA) probes immobilized on beads, for the detection of mtDNA point mutations and sequence-specific supercoiled plasmid DNA at the picomolar range. The probes are capable of invading the double-stranded circular DNA and forming a stable triplex structure. Thus, this method can significantly reduce the sample preparation and omit the PCR amplification steps prior to sensing. Further, this bioanalytical tool can open up a new paradigm in clinical settings for the screening of double-stranded circular nucleic acids with a single-base mismatch specificity in a rapid and sensitive manner.

Nucleic acid detection with CRISPR-Cas13a/C2c2.

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.