PepDRED: De Novo Peptide Design with Strong Binding Affinity for Target Protein.

De novo design of peptides that bind specifically to functional proteins is beneficial for diagnostics and therapeutics. However, complex permutations and combinations of amino acids pose significant challenges to the rational design of peptides with desirable stability and affinity. Herein, we develop a computational-based evolution method, namely, peptidomimetics-driven recognition elements design (PepDRED), to derive hemoglobin-inspired peptidomimetics. PepDRED mimics the natural evolutionism pipeline to generate stable apovariant (AVs) structures for wild-type counterparts via automated point mutations and validates their efficiency through free binding energy analysis and per residue energy decomposition analysis. For application demonstration, we applied PepDRED to design de novo peptides to bind FhuA, a typical TonB-dependent transporter (TBDT). TBDTs are Gram-negative bacterial outer membrane proteins responsible for iron transport and vital for bacterial resistance. PepDRED generated a pool of AVs and proceeded to reach an optimized peptide, AV440, with a remarkable binding affinity of -21 kcal/mol. AV440 is ∼2.5-fold stronger than the existing FhuA inhibitor Microcin J25. Network energy analysis further unveils that incorporating methionine (M42) in the N-terminal region significantly enhances inter-residue contacts and binding affinity. PepDRED offers a prompt and efficient in silico approach to develop potent peptide candidates for target proteins.

Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers.

Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-to-noise ratio compared to conventional antibody-based scWB. Being more time-saving (less than 25 min per cycle) and economical (1/1000 cost of conventional antibody probes), FAS-WB offers a highly efficient platform for profiling multiplex proteins at single-cell resolution.