An Investigation of Nirmatrelvir (Paxlovid) Resistance in SARS-CoV-2 M.

The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 M variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations.

High-Resolution Substrate Specificity Profiling of SARS-CoV-2 M; Comparison to SARS-CoV M.

The SARS-CoV-2 M protease from COVID-19 cleaves the pp1a and pp2b polyproteins at 11 sites during viral maturation and is the target of Nirmatrelvir, one of the two components of the frontline treatment sold as Paxlovid. We used the YESS 2.0 platform, combining protease and substrate expression in the yeast endoplasmic reticulum with fluorescence-activated cell sorting and next-generation sequencing, to carry out the high-resolution substrate specificity profiling of SARS-CoV-2 M as well as the related SARS-CoV M from SARS 2003.

Chemical and linguistic considerations for encoding Chinese characters: an embodiment using chain-end degradable sequence-defined oligourethanes created by consecutive solid phase click chemistry.

Sequence-defined polymers (SDPs) are currently being investigated for use as information storage media. As the number of monomers in the SDPs increases, with a corresponding increase in mathematical base, the use of tandem-MS for sequencing becomes more challenging. In contrast, chain-end degradation routines are truly , potentially allowing very large mathematical bases for encoding.

Aggregation of Charge Acceptors on Nanocrystal Surfaces Alters Rates of Photoinduced Electron Transfer.

Semiconductor nanocrystals (NCs) interfaced with molecular ligands that function as charge and energy acceptors are an emerging platform for the design of light-harvesting, photon-upconverting, and photocatalytic materials. However, NC systems explored for these applications often feature high concentrations of bound acceptor ligands, which can lead to ligand-ligand interactions that may alter each system's ability to undergo charge and energy transfer. Here, we demonstrate that aggregation of acceptor ligands impacts the rate of photoinduced NC-to-ligand charge transfer between lead(II) sulfide (PbS) NCs and perylenediimide (PDI) electron acceptors.

Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG) linkers at the peptide attachment points.

Using Spectator Ligands to Enhance Nanocrystal-to-Molecule Electron Transfer.

Semiconductor nanocrystals (NCs) have emerged as promising photocatalysts. However, NCs are often functionalized with complex ligand shells that contain not only charge acceptors but also other "spectator ligands" that control NC solubility and affinity for target reactants. Here, we show that spectator ligands are not passive observers of photoinduced charge transfer but rather play an active role in this process.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.

YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants.

Here we describe YESS 2.0, a highly versatile version of the yeast endoplasmic sequestration screening (YESS) system suitable for engineering and characterizing protein/peptide modifying enzymes such as proteases with desired new activities. By incorporating features that modulate gene transcription as well as substrate and enzyme spatial sequestration, YESS 2.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma.

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq ) to the spike ectodomain (S-ECD ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.

Mechanistic Analysis of Solid-State Colorimetric Switching: Monoalkoxynaphthalene-Naphthalimide Donor-Acceptor Dyads.

There is growing interest in creating solids that are responsive to various stimuli. Herein we report the first molecular-level mechanistic picture of the thermochromic polymorphic transition in a series of MAN-NI dyad crystals that turn from orange to yellow upon heating with minimal changes to the microscopic morphology following the transition. Detailed structural analyses revealed that the dyads assemble to create an alternating bilayer type structure, with horizontal alternating alkyl and stacked aromatic layers in both the orange and yellow forms.

Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression.

For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα.

Quantitative Analysis of the Substrate Specificity of Human Rhinovirus 3C Protease and Exploration of Its Substrate Recognition Mechanisms.

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in , a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1' positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1' position, respectively.

Rapid Screen for Tyrosine Kinase Inhibitor Resistance Mutations and Substrate Specificity.

We present a rapid and high-throughput yeast and flow cytometry based method for predicting kinase inhibitor resistance mutations and determining kinase peptide substrate specificity. Despite the widespread success of targeted kinase inhibitors as cancer therapeutics, resistance mutations arising within the kinase domain of an oncogenic target present a major impediment to sustained treatment efficacy. Our method, which is based on the previously reported YESS system, recapitulated all validated BCR-ABL1 mutations leading to clinical resistance to the second-generation inhibitor dasatinib, in addition to identifying numerous other mutations which have been previously observed in patients, but not yet validated as drivers of resistance.

Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of .

An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within cells, we performed comprehensive ERS analyses.

Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER.

Yeast Endoplasmic Reticulum Sequestration Screening for the Engineering of Proteases from Libraries Expressed in Yeast.

There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis.

Time-Dependent Solid State Polymorphism of a Series of Donor-Acceptor Dyads.

In order to exploit the use of favorable electrostatic interactions between aromatic units in directing the assembly of donor-acceptor (D-A) dyads, the present work examines the ability of conjugated aromatic D-A dyads with symmetric side chains to exhibit solid-state polymorphism as a function of time during the solid formation process. Four such dyads were synthesized and their packing in the solid-state from either slower (10-20 days) or faster (1-2 days) evaporation from solvent was investigated using single crystal X-ray analysis and powder X-ray diffraction. Two of the dyads exhibited tail-to-tail (A-A) packing upon slower evaporation from solvent and head-to-tail (D-A) packing upon faster evaporation from solvent.

NDI and DAN DNA: nucleic acid-directed assembly of NDI and DAN.

Two novel DNA base surrogate phosphoramidites 1 and 2, based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI), respectively, were designed, synthesized, and incorporated into DNA oligonucleotide strands. The DAN and NDI artificial DNA bases were inserted within a three-base-pair region within the interior of a 12-mer oligonucleotide duplex in various sequential arrangements and investigated with CD spectroscopy and UV melting curve analysis. The CD spectra of the modified duplexes indicated B-form DNA topology.

Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites.

The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound.

More than meets the eye: conformational switching of a stacked dialkoxynaphthalene-naphthalenetetracarboxylic diimide (DAN-NDI) foldamer to an NDI-NDI fibril aggregate.

The thermally induced conformational switching of a stacked dialkxoynaphthalene-naphthalenetetracarboxylic diimide (DAN-NDI) amphiphilic foldamer to an NDI-NDI fibril aggregate is described. The aggregated fibril structures were explored by UV/Vis, circular dichroism (CD), atomic-force microscopy (AFM), and TEM techniques. Our findings indicate that the aromatic DAN-NDI interactions of the original foldamer undergoes transformation to a fibrillar assembly with aromatic NDI-NDI stacked interactions.

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.

Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library.

Commercial proteases: present and future.

This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering.

Increased antibody affinity confers broad in vitro protection against escape mutants of severe acute respiratory syndrome coronavirus.

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (K(D)) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV.

Reactions of brominated naphthalene diimide with bis(tributylstannyl)acetylene: a simple approach for conjugated polymers and versatile coupling intermediates.

A new synthetic approach to 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) containing materials and conjugates is described. A simple one-step Stille coupling procedure is used to create either novel alkyne-linked NDI polymers or a new stannylated diyne synthetic building block that provides a flexible approach to new NDI conjugates and polymers.

Subtle recognition of 14-base pair DNA sequences via threading polyintercalation.

Small molecules that bind DNA in a sequence-specific manner could act as antibiotic, antiviral, or anticancer agents because of their potential ability to manipulate gene expression. Our laboratory has developed threading polyintercalators based on 1,4,5,8-naphthalene diimide (NDI) units connected in a head-to-tail fashion by flexible peptide linkers. Previously, a threading tetraintercalator composed of alternating minor-major-minor groove-binding modules was shown to bind specifically to a 14 bp DNA sequence with a dissociation half-life of 16 days [Holman, G.