· Direct sequencing of 2’-deoxy-2’- fluoroarabinonucleic acid (FANA) using nanopore- induced phase-shift sequencing (NIPSS)

Person in charge: Li Xintong

 2’-deoxy-2’-fluoroarabinonucleic acid (FANA), which is one type of xeno-nucleic acid (XNA), has been intensively studied in molecular medicine and synthetic biology because of its superior gene-silencing and catalytic activities. Although urgently required, FANA cannot be directly sequenced by any existing platform. Nanopore sequencing, which identifies a single molecule analyte directly from its physical and chemical properties, shows promise for direct XNA sequencing. As a proof of concept, different FANA homopolymers show well-distinguished pore blockage signals in a Mycobacterium smegmatis porin A (MspA) nanopore. By ligating FANA with a DNA drive-strand, direct FANA sequencing has been demonstrated using phi29 DNA polymerase by Nanopore-Induced Phase Shift Sequencing (NIPSS). When bound with an FANA template, the phi29 DNA polymerase shows unexpected reverse transcriptase activity when monitored in a single molecule assay. Following further investigations into the ensemble, phi29 DNA polymerase is shown to be a previously unknown reverse transcriptase for FANA that operates at room temperature, and is potentially ideal for nanopore sequencing. These results represent the first direct sequencing of a sugar-modified XNA and suggest that phi29 DNA polymerase could act as a promising enzyme for sustained sequencing of a wide variety of XNAs

· Aptamer-based Western blot for selective protein recognition

Person in charge: Wang Yao

 Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of antibody, which often brings high production cost and requires long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition. And replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP and His-tag. The generated aptamers G1, M1 and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.

Aptamers for protein selective recognition in Western blot

· Study on enhancing the activity of functional nucleic acids by artificial modification

Person in charge: Yang Jintao

    Functional nucleic acids include aptamers with specific affinity and nucleases with catalytic activity. One important reason for the relatively limited application scenarios of functional nucleic acids is that there are few types of chemical functional groups in nucleic acid molecules.To solve this problem, this project develops biological orthogonal reaction, introduces artificial modification on nucleic acid, explores the law of artificial modification affecting the activity of functional nucleic acid, and develops methods of artificial modification improving the activity of functional nucleic acid.

Site directed modification can provide DNA molecular catalytic activity

· Alternative RNA splicing catalyzed by deoxyribozymes

Person in charge: Wei Dongying

 Introns and their removal play fundamental roles in life processes but remain difficult to achieve in in vitro molecular experiments. Here, we developed a DNAzymes-based RNA in vitro splicing method. It can be completed under simple experimental conditions in one system within 4 hours without purification during the process.We first completed the splicing and alternative splicing of the conceptual RNA. This method was then used to splicing two different functional RNA, the mRNA of CTLA-4 (Homo sapiens), and the type III hammerhead ribozyme (sTRSV).

RNA splicing system catalyzed by deoxyribozyme