The inherent specificity of DNA sequence hybridization has been extensively exploited to develop bioengineering applications. Nevertheless, the structural potential of DNA has been far less explored for creating non-canonical DNA-based reactions. Here we develop a DNA origami-enabled highly localized metallization reaction for intrinsic metallization patterning with 10-nm resolution. Both theoretical and experimental studies reveal that low-valence metal ions (Cu2+ and Ag+) strongly coordinate with DNA bases in protruding clustered DNA (pcDNA) prescribed on two-dimensional DNA origami, which results in effective attraction within flexible pcDNA strands for site-specific pcDNA condensation. We find that the metallization reactions occur selectively on prescribed sites while not on origami substrates. This strategy is generically applicable for free-style metal painting of alphabet letters, digits and geometric shapes on all−DNA substrates with near-unity efficiency. We have further fabricated single- and double-layer nanoscale printed circuit board (nano-PCB) mimics, shedding light on bio-inspired fabrication for nanoelectronic and nanophotonic applications.

Coronavirus 3C-like protease (3CL Pro ) is a highly conserved cysteine protease employing a catalytic dyad for its functions. 3CL Pro is essential to the viral life cycle and, therefore, is an attractive target for developing antiviral agents. However, the detailed catalytic mechanism of coronavirus 3CL Pro remains largely unknown. We took an integrated approach of employing X-ray crystallography, mutational studies, enzyme kinetics study, and inhibitors to gain insights into the mechanism. Such experimental work is supplemented by computational studies, including the prereaction state analysis, the ab initio calculation of the critical catalytic step, and the molecular dynamic simulation of the wild-type and mutant enzymes. Taken together, such studies allowed us to identify a residue pair (Glu-His) and a conserved His as critical for binding; a conserved GSCGS motif as important for the start of catalysis, a partial negative charge cluster (PNCC) formed by Arg-Tyr-Asp as essential for catalysis, and a conserved water molecule mediating the remote interaction between PNCC and catalytic dyad. The data collected and our insights into the detailed mechanism have allowed us to achieve a good understanding of the difference in catalytic efficiency between 3CL Pro from SARS and MERS, conduct mutational studies to improve the catalytic activity by 8-fold, optimize existing inhibitors to improve the potency by 4-fold, and identify a potential allosteric site for inhibitor design. All such results reinforce each other to support the overall catalytic mechanism proposed herein.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Reconfiguration of membrane protein channels for gated transport is highly regulated under physiological conditions. However, a mechanistic understanding of such channels remains challenging owing to the difficulty in probing subtle gating-associated structural changes. Herein, we show that charge neutralization can drive the shape reconfiguration of a biomimetic 6-helix bundle DNA nanotube (6HB). Specifically, 6HB adopts a compact state when its charge is neutralized by Mg2+; whereas Na+ switches it to the expanded state, as revealed by MD simulations, small-angle X-ray scattering (SAXS), and FRET characterization. Furthermore, partial neutralization of the DNA backbone charges by chemical modification renders 6HB compact and insensitive to ions, suggesting an interplay between electrostatic and hydrophobic forces in the channels. This system provides a platform for understanding the structure-function relationship of biological channels and designing rules for the shape control of DNA nanostructures in biomedical applications.

Enterovirus 71 (EV71) is the causative agent of hand, foot, and mouth disease (HFMD), which typically affects infants and children (1). HFMD usually presents as a mild febrile disease with a localized rash, but some patients may develop infection of the central nervous system (CNS) with illness ranging from aseptic meningitis through fatal encephalitis (2). In the last decade, outbreaks of HFMD have regularly reoccurred through Asia (3, 4). According to data from the Chinese Center for Disease Control and Prevention (CDC), more than 420,000 cases of HFMD, with 70 deaths, were reported in China in April 2014. Currently, there is no antiviral therapy available for treatment of HFMD.EV71 belongs to the genus Enterovirus in the family Picornaviridae (5-7). Similar to other picornaviruses, EV71 contains a single-stranded, positive-sense RNA encoding a large polyprotein precursor (8, 9). The polyprotein is further cleaved into four structural proteins (VP1 to VP4) to form the viral capsid and seven nonstructural proteins (2A to 3D) for virus replication via the 2A protease and 3C protease (10, 11). Except for the cleavage of VP1/2A by the 2A protease (12) and the RNA-dependent cleavage of VP2/4 (13), the 3C protease is absolutely required for the cleavage of other junction sites within the polyprotein (14-16). Meanwhile, EV71 3C reportedly interferes with the polyadenylation of host cell RNA by digesting CstF-64, a critical host factor for 3= pre-mRNA processing, suggesting a novel mechanism by which picornaviruses utilize 3Cpro to impair host cell function (17). In addition, the 3C protease can also cleave numerous factors and regulators associated with cellular DNA-dependent RNA polymerases I, II, and III, such as the octamer-binding protein (OCT-1), TATA box-binding protein (TBP), cyclic AMP-responsive element-binding protein (CREB), transcription activator p53, histone H3, and DNA polymerase III (18-21). The pivotal role of 3C protease in EV71 replication makes it an attractive target for antiviral discovery (22).The crystal structure of unliganded EV71 3C protease showed that EV71 3C protease folded into two domains that are related to other picornaviral 3C protease structures (23). The complex structures of EV71 mutants H133G, E71A, E71D with the inhibitor rupintrivir are similar to that of the unliganded protease structure (24). Lu et al. thoroughly characterized the 3C proteases from EV71 and CVA16 and reported a series of structures of both enzymes in free, peptide-bound, or inhibitor-bound form (25). These findings provided precise molecular insights into the substrate recognition and inhibition of 3C protease.