Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

<p>Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - the peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.<i></i></p>

Rapid discovery and development of serum-stable, selective, and high affinity peptide-based binders to protein targets are challenging. Angiotensin converting enzyme 2 (ACE2) has recently been identified as a cardiovascular disease biomarker and the primary receptor utilized by the severe acute respiratory syndrome coronavirus 2. In this study, we report the discovery of high affinity peptidomimetic binders to ACE2 via affinity selection-mass spectrometry (AS-MS). Multiple high affinity ACE2-binding peptides (ABP) were identified by selection from canonical and noncanonical peptidomimetic libraries containing 200 million members (dissociation constant, KD = 19–123 nM). The most potent noncanonical ACE2 peptide binder, ABP N1 (KD = 19 nM), showed enhanced serum stability in comparison with the most potent canonical binder, ABP C7 (KD = 26 nM). Picomolar to low nanomolar ACE2 concentrations in human serum were detected selectively using ABP N1 in an enzyme-linked immunosorbent assay. The discovery of serum-stable noncanonical peptidomimetics like ABP N1 from a single-pass selection demonstrates the utility of advanced AS-MS for accelerated development of affinity reagents to protein targets.

<p>Ribosomes produce most proteins of living cells in seconds. Here we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids over 328 consecutive reactions. The machine is rapid - the peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.<i></i></p>

MicroRNAs (miRNAs) are implicated in the onset and progression of a variety of diseases. Modulating the expression of specific miRNAs is a possible option for therapeutic intervention. A promising strategy is the use of antisense oligonucleotides (ASOs) to inhibit miRNAs. Targeting ASOs to specific tissues can potentially lower the dosage and improve clinical outcomes by alleviating systemic toxicity. We leverage here automated peptide nucleic acid (PNA) synthesis technology to manufacture an anti-miRNA oligonucleotide (antagomir) covalently attached to a 12-mer peptide that binds to transferrin receptor 1. Our PNA-peptide conjugate is active in cells and animals, effectively inhibiting the expression of miRNA-21 both in cultured mouse cardiomyocytes and different mouse organs (heart, liver, kidney, lung, and spleen), while remaining well-tolerated in animals up to the highest tested dose of 30 mg/kg. Conjugating the targeting ligand to the PNA antagomir significantly improved inhibition of miRNA-21 in the heart by over 50% relative to the PNA alone. Given the modulation of biodistribution observed with our PNA-peptide conjugate, we anticipate this antagomir platform to serve as a starting point for pre-clinical development studies.Table of Contents EntrySynopsisConjugating T12, a peptide targeting transferrin receptor 1 (TfR1), to a peptide nucleic acid (PNA) oligonucleotide targeting microRNA-21 increases delivery of the PNA-T12 conjugate to cardiac tissue relative to PNA alone.