Recombinant Fusion Streptavidin as a Scaffold for DNA Nanotetrads for Nucleic Acid Delivery and Telomerase Activity Imaging in Living Cells.

Efficient platforms for intracellular delivery of nucleic acids are essential for biomedical imaging and gene regulation. We develop a recombinant fusion streptavidin as a novel protein scaffold for DNA nanotetrads for highly efficient nucleic acid delivery and telomerase activity imaging in living cells via cross-linking hybridization chain reaction (cHCR). The recombinant streptavidin protein is designed to fuse with multiple SV40 NLS (nuclear localization signal) and NES (nuclear export signal) domains and prepared through Escherichia coli expression. The recombinant NLS-SA protein allows facile assembly with four biotinylated DNA probes via high-affinity noncovalent interactions, forming a well-defined DNA tetrad nanostructure. The DNA nanotetrads are demonstrated to confer efficient cytosolic delivery of nucleic acid via a caveolar mediated endocytosis pathway, allowing efficient escape from lysosomal degradation. Moreover, the nanotetrads enable efficient cHCR assembly in response to telomerase in vitro and in cellulo, affording ultrasensitive detection and spatially resolved imaging for telomerase with a detection limit as low as 90 HeLa cells/mL. The fluorescence brightness obtained in live cell imaging is found to be dynamically correlated to telomerase activity and the inhibitor concentrations. Therefore, the proposed strategy may provide a highly efficient platform for nucleic acid delivery and imaging of biomarkers in living cells.

Computational study of the kinetics and mechanisms for the HCO + O3 reaction.

The mechanisms of radical-molecule reactions between HCO (formyl radical) and O3 (ozone) have been investigated by using BH&HLYP and QCISD methods with the 6-311++G(3df,2p) basis set. The energetics have been refined with CCSD(T) and QCISD(T) theoretical approaches with the same basis set based on the geometries calculated at the QCISD method. The intermediates of hydrogen-bonded complexes and the critical transition states are also examined with the multireference methods. Two possible reaction pathways containing hydrogen-abstraction and association-elimination processes for the interaction of HCO with O3 are proposed. Both reaction mechanisms can occur via the prereactive hydrogen-bonded complex, O3-HCO, with 2.45 kcal/mol stability at the CCSD(T) approach with respect to the reactants; even so, the hydrogen-abstraction mechanism exhibits a lower energy barrier. The rate constants for both processes are also predicted. The total rate constant at 298 K is calculated to be in close agreement with the experimental value of 8.3 × 10(-13) cm(3) molecule(-1) s(-1).