Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the tens of ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.As the vaccine deployment starts worldwide for COVID-19, broad antibody testing for SARS-CoV-2 faces severe limitations. Although nucleic acid testing is critical to detecting the virus, serological antibody tests are vital tools for monitoring the dynamic human humoral response to SARS-CoV-2 vaccination and viral infection 1 . Population-scale, longitudinal evaluation of antibody responses is needed to determine the strength and duration of immunity to the primary virus, to the variants, and to vaccines, which is important in informing public policy and vaccination strategies 2-7 . In addition, antibody tests serve as a complement or an alternative to nucleic acid diagnostics for patients with a low viral load or for low-resource areas where expensive reverse transcription polymerase chain reaction (RT-PCR) testing is difficult to access [8][9][10] . Serological tests also support therapeutic development through identification of either individuals who could serve as donors for convalescent serum therapeutics 11 , or patients with potentially strong neutralizing antibodies that can be produced in vitro as new antivirals and prophylactics 12,13 . All these applications would be greatly accelerated with an assay that is simple, rapid and high throughput, without sacrificing accuracy and sensitivity.Traditional serological assays are not optimal in the face of this broad pandemic. The most widely used laboratory serological tests take the form of enzyme-linked immunosorbent assays