Xenoprotein engineering via synthetic libraries.

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ∼150-kDa protein target, a task that would be difficult or impossible by other means.

Conformational Dynamics in Extended RGD-Containing Peptides.

RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

Conformational Stabilization and Rapid Labeling of a 29-Residue Peptide by a Small Molecule Reaction Partner.

A 29-residue peptide (MP01), identified by in vitro selection for reactivity with a small molecule perfluoroaromatic, was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six-amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M-1 s-1 at pH 7.4 and room temperature, approximately 2 orders of magnitude greater than that of its progenitor sequence and 3 orders of magnitude greater than background cysteine reactivity. MP01-Gen4 appeared to be conformationally dynamic and exhibited several properties reminiscent of larger protein molecules, including denaturant-sensitive structure and reactivity. We believe the majority of the reaction rate enhancement can be attributed to interaction of MP01-Gen4 with the perfluoroaromatic probe, which was found to stabilize a helical conformation of both MP01-Gen4 and nonreactive Cys-to-Ser or Cys-to-Ala variants. These findings demonstrate the ability of dynamic peptides to access proteinlike reaction mechanisms and the potential of perfluoroaromatic functionality to stabilize small peptide folds.

A fully automated flow-based approach for accelerated peptide synthesis.

Here we report a fully automated, flow-based approach to solid-phase polypeptide synthesis, with amide bond formation in 7 seconds and total synthesis times of 40 seconds per amino acid residue. Crude peptide purities and isolated yields were comparable to those for standard-batch solid-phase peptide synthesis. At full capacity, this approach can yield tens of thousands of individual 30-mer peptides per year.

Antibody-Bactericidal Macrocyclic Peptide Conjugates To Target Gram-Negative Bacteria.

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off-target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody-bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram-negative bacteria. We used cysteine SN Ar chemistry to synthesize and systematically study a library of large (>30-mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.

Reversible changes in solution pH resulting from changes in thermoresponsive polymer solubility.

Pendant groups on polymers that have lower-critical solution temperature (LCST) properties experience a water-like environment below the LCST where the polymer is soluble but are less hydrated above the LCST when the polymer phase separates from solution. When these pendant groups are amphoteric groups like carboxylate salts or ammonium salts, the change in solvation that accompanies the polymer precipitation event significantly changes these groups' acidity or basicity. These changes in acidity or basicity can lead to carboxylate salts forming carboxylic acid groups by capturing protons from the bulk solvent or ammonium salts reverting to the neutral amine by release of protons to the bulk solvent, respectively. When polymers like poly(N-isopropylacrylamide) that contain a sufficient loading of such comonomers are dissolved in solutions whose pH is near the pK(a) of the pendant acid or basic group and undergo an LCST event, the LCST event can change the bulk solution pH. These changes are reversible. These effects were visually followed using common indicators with soluble polymers and or by monitoring solution pH as a function of temperature. LCST events triggered by the addition of a kosmotropic salt lead to similar reversible solution pH changes.

Automated Flow Synthesis of Peptide-PNA Conjugates.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5' untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 µM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results.

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics. IMPORTANCE To meet the challenges imposed by the COVID-19 pandemic, research laboratories shifted their focus and clinical diagnostic laboratories developed and utilized new assays. Nucleic acid-based testing became widespread and, for the first time, was used as a prophylactic measure. We report 15 cases of researchers at two institutes testing positive for SARS-CoV-2 on routine surveillance tests, in the absence of any symptoms or transmission. These researchers were likely contaminated with nonhazardous nucleic acids generated in the laboratory in the course of developing new SARS-CoV-2 diagnostics. These contaminating nucleic acids were persistent and widespread throughout the laboratory. We report these findings as a cautionary tale to those working with nucleic acids used in diagnostic testing and as a call for careful stewardship of diagnostically relevant molecules. Our conclusions are especially relevant as at-home COVID-19 testing gains traction in the marketplace and these amplicons may impact on the general public.

Quantifying residue-specific conformational dynamics of a highly reactive 29-mer peptide.

Understanding structural transitions within macromolecules remains an important challenge in biochemistry, with important implications for drug development and medicine. Insight into molecular behavior often requires residue-specific dynamics measurement at micromolar concentrations. We studied MP01-Gen4, a library peptide selected to rapidly undergo bioconjugation, by using electron paramagnetic resonance (EPR) to measure conformational dynamics. We mapped the dynamics of MP01-Gen4 with residue-specificity and identified the regions involved in a structural transformation related to the conjugation reaction. Upon reaction, the conformational dynamics of residues near the termini slow significantly more than central residues, indicating that the reaction induces a structural transition far from the reaction site. Arrhenius analysis demonstrates a nearly threefold decrease in the activation energy of conformational diffusion upon reaction (8.0 kBT to 3.4 kBT), which occurs across the entire peptide, independently of residue position. This novel approach to EPR spectral analysis provides insight into the positional extent of disorder and the nature of the energy landscape of a highly reactive, intrinsically disordered library peptide before and after conjugation.

A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins.

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.