Chemically modified DNAzyme with enhanced activity for sensitive microRNA imaging in live cells
Person in charge: Chen Jiawen
Source link: https://doi.org/10.3390/molecules31081271
As critical regulators of gene expression, microRNAs (miRNAs) are key biomarkers and therapeutic targets in cancer. However, current methods for intracellular miRNA imaging are often limited by poor sensitivity and operational complexity. In this study, we identified a site-specifically modified DNAzyme variant, 11Bn, which exhibits up to 7-fold higher catalytic activity than the wild-type 8-17 through systematic screening. Using this variant, we constructed a DNAzyme-based sensor for miRNA-21 imaging in living cells. The sensor achieves a limit of detection of 7.89 nM, threefold lower than that of the wild-type sensor, and enables sensitive visualization of intracellular miRNA-21 without signal amplification. Moreover, it can capture dynamic changes in miRNA levels within cells, providing a versatile molecular tool for miRNA imaging and related biomedical applications.
Chemically modified DNAzyme-based sensor for miRNA imaging
Sanger sequencing of xeno-nucleic acid
Person in charge: Wang Yueyao
Source link: https://doi.org/10.1002/anie.202516617
Xeno-nucleic acids (XNAs) offer biostable genetic polymers for biotechnology and information technology, but are incompatible with prevailing DNA sequencing methodologies. Here we report a Sanger sequencing approach tailored for XNA, enabling direct readout of XNA strands up to 50 bases. This new method relies on a Bst DNA polymerase mutant, which not only recognizes XNA as a template but also efficiently incorporates dideoxyribonucleoside triphosphate (ddNTP) substrates, facilitating sequencing of XNA by synthesis of complementary DNA. The Bst F710Y mutant exhibits at least two orders of magnitude higher activity in catalyzing ddNTP incorporation. Our method demonstrates accurate sequence determination of diverse XNA strands of distinct backbone chemistries such as TNA and FANA. Lastly, we showcase proof-of-concept automated XNA sequencing on a genetic analyzer instrument, by leveraging Bst mutant's ability to stochastically incorporate BigDye-labeled ddNTP substrates in a single reaction. This work establishes a direct sequencing platform for XNA employing a chain termination strategy, and lays a foundation for future de novo identification of functional XNA and development of XNA-based data storage system.
Sanger sequencing method for XNA
Chemoenzymatic installation of site-specific chemical groups on DNA enhances the catalytic activity
Person in charge: Zhang Ze, Chen Siqi, Yang Jintao
Source link: https://doi.org/10.1021/jacs.4c00484
Functional DNAs are valuable molecular tools in chemical biology and analytical chemistry but suffer from low activities due to their limited chemical functionalities. Here, we present a chemoenzymatic method for site-specific installation of diverse functional groups on DNA, and showcase the application of this method to enhance the catalytic activity of a DNA catalyst. Through chemoenzymatic introduction of distinct chemical groups, such as hydroxyl, carboxyl, and benzyl, at specific positions, we achieve significant enhancements in the catalytic activity of the RNA-cleaving deoxyribozyme 10–23. A single carboxyl modification results in a 100-fold increase, while dual modifications (carboxyl and benzyl) yield an approximately 700-fold increase in activity when an RNA cleavage reaction is catalyzed on a DNA–RNA chimeric substrate. The resulting dually modified DNA catalyst, CaBn, exhibits a kobs of 3.76 min-1 in the presence of 1 mM Mg2+ and can be employed for fluorescent imaging of intracellular magnesium ions. Molecular dynamics simulations reveal the superior capability of CaBn to recruit magnesium ions to metal-ion-binding site 2 and adopt a catalytically competent conformation. Our work provides a broadly accessible strategy for DNA functionalization with diverse chemical modifications, and CaBn offers a highly active DNA catalyst with immense potential in chemistry and biotechnology.
Facile chemoenzymatic method for site-specific DNA modifications
Enzymatic synthesis of TNA protects DNA nanostructures
Person in charge: Qin Bohe
Source link: https://doi.org/10.1002/anie.202317334
Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3’ terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.
TNA extension protects DNAs from nuclease digestion
Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)
Person in charge: Li Xintong
Source link: https://doi.org/10.1039/c8sc05228j
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.
Direct sequencing of FANA using Nanopore-Induced Phase-Shift Sequencing (NIPSS)
