Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses.

Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.

Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations.

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors.

Identification of binding specificity-determining features in protein families.

We present a new approach for identifying features of ligand-protein binding interfaces that predict binding selectivity and demonstrate its effectiveness for predicting kinase inhibitor specificity. We analyzed a large set of human kinases and kinase inhibitors using clustering of experimentally determined inhibition constants (to define specificity classes of kinases and inhibitors) and virtual ligand docking (to extract structural and chemical features of the ligand-protein binding interfaces). We then used statistical methods to identify features characteristic of each class.

SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2'-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 base pairs) rendered primer extension impractical.

Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B.

During (-)-strand DNA synthesis in retroviruses and Saccharomyces cerevisiae LTR retrotransposons, a purine rich region of the RNA template, known as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNA-dependent DNA synthesis. Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include runs of purine ribonucleotides, it has been suggested that these PPTs are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism. Here, we have used the aminoglycoside neomycin B (NB) to examine which structural features of the Ty3 PPT contribute to specific recognition and processing by its cognate RT.

Noncovalent probes for the investigation of structure and dynamics of protein-nucleic acid assemblies: the case of NC-mediated dimerization of genomic RNA in HIV-1.

The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as noncovalent probes. Small-molecule ligands make contact with their target substrates through complex combinations of H-bonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies.

Like polarity ion/ion reactions enable the investigation of specific metal interactions in nucleic acids and their noncovalent assemblies.

A rare example of ion/ion reaction between species of like polarity was shown to take place during the transfer of metal cations from nucleic acid substrates to chelating agents in the gas phase. Gaseous anionic reactants were generated from separate solutions of analyte and chelator by using a dual nanospray setup. The respective multiply charged ions shared the same path and were allowed to react for a predetermined interval in an rf-only hexapole before high-resolution analysis by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry.

Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element.

Nuclear export of certain HIV-1 mRNAs requires an interaction between the viral Rev protein and the Rev response element (RRE), a structured element located in the Env region of its RNA genome. This interaction is an attractive target for both drug design and gene therapy, exemplified by RevM10, a transdominant negative protein that, when introduced into host cells, disrupts viral mRNA export. However, two silent G->A mutations in the RRE (RRE61) confer RevM10 resistance, which prompted us to examine RRE structure using a novel chemical probing strategy.

Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction.

Measuring the binding stoichiometry of HIV-1 Gag to very-low-density oligonucleotide surfaces using surface plasmon resonance spectroscopy.

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions.

End-stacking of copper cationic porphyrins on parallel-stranded guanine quadruplexes.

Nucleic acids that contain multiple sequential guanines assemble into guanine quadruplexes (G-quadruplexes). Drugs that induce or stabilize G-quadruplexes are of interest because of their potential use as therapeutics. Previously, we reported on the interaction of the Cu(2+) derivative of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine (CuTMpyP4), with the parallel-stranded G-quadruplexes formed by d(T(4)G( n )T(4)) (n = 4 or 8) (Keating and Szalai in Biochemistry 43:15891-15900, 2004).

Understanding the isomerization of the HIV-1 dimerization initiation domain by the nucleocapsid protein.

The specific binding of HIV-1 nucleocapsid (NC) to the hinge region of the kissing-loop (KL) dimer formed by stemloop 1 (SL1) can have significant consequences on its ability to isomerize into the corresponding extended duplex (ED) form. The binding determinants and the effects on the isomerization process were investigated in vitro by a concerted strategy involving ad hoc RNA mutants and electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry, which enabled us to characterize the stoichiometry and conformational state of all possible protein-RNA and RNA-RNA assemblies present simultaneously in solution. For the first time, NC-hinge interactions were observed in constructs including at least one unpaired guanine at the 5'-end of the loop-loop duplex, whereas no interactions were detected when the unpaired guanine was placed at its 3'-end.

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry.

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (Psi-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the Psi-RNA domains.

Inhibitory effects of archetypical nucleic acid ligands on the interactions of HIV-1 nucleocapsid protein with elements of Psi-RNA.

Disrupting the interactions between human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein and structural elements of the packaging signal (Psi-RNA) could constitute an ideal strategy to inhibit the functions of this region of the genome leader in the virus life cycle. We have employed electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS) to assess the ability of a series of nucleic acid ligands to bind selected structures of Psi-RNA and inhibit their specific interactions with NC in vitro. We found that the majority of the ligands included in the study were able to form stable non-covalent complexes with stem-loop 2, 3 and 4 (SL2-4), consistent with their characteristic nucleic acid binding modes.

Identification of a novel non-carbohydrate molecule that binds to the ribosomal A-site RNA.

We report the identification of a novel compound that binds to the Escherichia coli 16S ribosomal A-site. Binding by the compound was observed using nuclear magnetic resonance and mass spectrometry techniques. We show that the compound binds in the same position in the A-site RNA as occupied by the aminoglycoside class of antibiotics.

cADPR analogues: effect of an adenosine 2'- or 3'-methoxy group on conformation.

[structure: see text] The 2'-OMe-A (2) and 3'-OMe-A (3) analogues of the calcium release agent cADPR (1) were prepared and their solution structures studied by NMR spectroscopy. Compared to 1, 2 shows a shift in its A ring conformation and changes in its R ring N:S and gammat:gamma+ ratios, while 3 displays a significant change in the conformation of its A ring gamma-bond.