Yongyun Zhao

Cleavage-based signal amplification of RNA

RNA detection has become an integral part of current biomedical research. Up to now, the reverse transcription-PCR has been the most practical method to detect mRNA targets. However, RNA detection by reverse transcription-PCR requires sophisticated equipment and it is highly sensitive to contamination with genomic DNA. Here we report a new isothermal reaction to simultaneously amplify and detect RNA, based on cleavage by DNAzyme and signal amplification. Cleavage-based signal amplification of RNA cannot be contaminated by genomic DNA and is suitable for the detection of both mRNA and microRNA targets, with high specificity and sensitivity. Moreover, the detection results can be reported in a colorimetric or real-time fluorometric way for different detection purposes.

Tumor cell characterization and classification based on cellular specific membrane capacitance and cytoplasm conductivity.

This paper reports a microfluidic system that enables the characterization of tumor cell electrical properties where cells were aspirated through a constriction channel (cross-section area smaller than that of biological cells) with cellular impedance profiles measured and translated to specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm). Two batches of H1299 cells were quantified by the microfluidic platform with different constriction channel cross-section areas, recording no differences with statistical significance (p<0.001) in both Cspecific membrane (1.63±0.52 vs. 1.65±0.43 μF/cm(2)) and σcytoplasm (0.90±0.19 vs. 0.92±0.15S/m), and thus confirming the reliability of the microfluidic platform. For paired high- and low-metastatic carcinoma strains 95D (ncell=537) and 95C cells (ncell=486), significant differences in both Cspecific membrane (2.00±0.43 vs. 1.62±0.39 μF/cm(2)) and σcytoplasm (0.88±0.46 vs. 1.25±0.35S/m) were observed. Statistically significant difference only in Cspecific membrane (2.00±0.43 vs. 1.58±0.30 μF/cm(2)) was observed for 95D cells (ncell=537) and 95D CCNY-KD cells with single oncogene CCNY down regulation (ncell=479, CCNY is a membrane-associated protein). In addition, statistically significant difference only in σcytoplasm (0.73±0.17 vs. 1.01±0.17S/m) was observed for A549 cells (ncell=487) and A549 CypA-KD cells with single oncogene CypA down regulation (ncell=597, CypA is a cytosolic protein). These results validated the developed microfluidic platform for Cspecific membrane and σcytoplasm quantification and confirmed the feasibility of using Cspecific membrane and σcytoplasm for tumor cell classification.

DNAzyme based gap-LCR detection of single-nucleotide polymorphism.

Fast and accurate detection of single-nucleotide polymorphism (SNP) is thought more and more important for understanding of human physiology and elucidating the molecular based diseases. A great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. However most of those methods developed to date incorporate complicated probe labeling and depend on advanced equipment. The DNAzyme based Gap-LCR detection method averts any chemical modification on probes and circumvents those problems by incorporating a short functional DNA sequence into one of LCR primers. Two kinds of exonuclease are utilized in our strategy to digest all the unreacted probes and release the DNAzymes embedded in the LCR product. The DNAzyme applied in our method is a versatile tool to report the result of SNP detection in colorimetric or fluorometric ways for different detection purposes.

In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity

Single-nucleotide polymorphisms, either inherited or due to spontaneous DNA damage, are associated with numerous diseases. Developing tools for site-specific nucleotide modification may one day provide a way to alter disease polymorphisms. Here, we describe the in vitro selection and characterization of a new deoxyribozyme called F-8, which catalyzes nucleotide excision specifically at thymidine. Cleavage by F-8 generates 3′- and 5′-phosphate ends recognized by DNA modifying enzymes, which repair the targeted deoxyribonucleotide while maintaining the integrity of the rest of the sequence. These results illustrate the potential of DNAzymes as tools for DNA manipulation.

Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin.

Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

Signal amplification of glucosamine-6-phosphate based on ribozyme glmS.

Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.

Selective tumor cell death induced by irradiated riboflavin through recognizing DNA G–T mismatch

Abstract Riboflavin (vitamin B2) has been thought to be a promising antitumoral agent in photodynamic therapy, though the further application of the method was limited by the unclear molecular mechanism. Our work reveals that riboflavin was able to recognize G–T mismatch specifically and induce single-strand breaks in duplex DNA targets efficiently under irradiation. In the presence of riboflavin, the photo-irradiation could induce the death of tumor cells that are defective in mismatch repair system selectively, highlighting the G–T mismatch as potential drug target for tumor cells. Moreover, riboflavin is a promising leading compound for further drug design due to its inherent specific recognition of the G–T mismatch.