Rapid discovery of self-assembling peptides with one-bead one-compound peptide library

Self-assembling peptides hold significant promise in materials science, nanoscience, and medicine. However, identifying self-assembling sequences from the vast combinatorial space of even short peptides remains a challenge. Traditional methods, such as empirical design based on known self-assembling peptides (e.g., KLVFF from amyloid-β, NFGAIL from islet amyloid polypeptide), computational screening of peptide sequences, and dynamic combinatorial libraries have limitations in terms of speed and unbiased discovery. This research explores the potential of the one-bead one-compound (OBOC) combinatorial library method to efficiently screen a large array of peptide sequences for self-assembly properties. The OBOC technique, known for its ability to screen vast peptide sequence spaces, is adapted here by incorporating a fluorescent probe sensitive to hydrophobic environments. This approach aims to overcome existing limitations by providing a faster and less biased method to discover novel self-assembling peptides. The ability to rapidly identify such peptides is crucial to accelerate development of functional nanomaterials for various applications. The choice of eukaryotic L-amino acids is motivated by their inherent biodegradability and biocompatibility, making them suitable for in vivo applications.

Existing methods for identifying self-assembling peptides involve empirical design, computational screening, and dynamic combinatorial library approaches. Empirical design relies on mimicking sequences from naturally occurring self-assembling peptides like KLVFF and NFGAIL, limiting diversity. Computational methods are useful for identifying candidate peptides but can be computationally intensive. Dynamic combinatorial libraries, while offering more diversity, involve complex enzymatic processes. Each approach has limitations in either the diversity of peptides screened or the efficiency of the screening process. The OBOC approach, previously used for identifying ligands and other bioactive peptides, provides a platform to overcome these challenges by combining a vast peptide library with a straightforward screening assay.

The researchers developed a fluorescent-activation screening assay based on the OBOC method. A random pentapeptide library (excluding cysteine) was synthesized on TentaGel S resin beads, with each bead carrying a unique peptide sequence. The N-terminus of each peptide was capped with nitro-1,2,3-benzoxadiazole (NBD), a fluorophore sensitive to hydrophobic environments. Self-assembly of the peptides on the beads creates hydrophobic pockets, leading to increased NBD fluorescence in an aqueous environment. The library was screened by visually identifying fluorescent beads under a microscope. Positive beads were isolated for sequencing using automated Edman degradation. The identified self-assembling peptide sequences were then resynthesized in soluble form, purified by HPLC, and characterized. The critical micelle concentration (CMC) of the peptides was determined to assess their self-assembly propensity. The morphology of the self-assembled structures was examined using transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy, UV-Vis spectroscopy, X-ray diffraction, and selected-area electron diffraction (SAED) were employed for structural characterization. The interaction of the identified self-assembling peptides with HeLa cells was assessed using confocal laser scanning microscopy (CLSM), including experiments evaluating cell uptake mechanisms and intracellular distribution. Cell viability assays were performed to evaluate the toxicity of the peptides.

Using the developed fluorescent-activation screening assay, the researchers screened approximately 100,000 beads from a random pentapeptide OBOC library and identified eight self-assembling pentapeptides: FTISD, ITSVV, YFTEF, ISDNL, LDFPI, FAGFT, FGFDP, and FFVDF. These peptides exhibited CMC values ranging from 8.4 to 14.8 µM, indicating a clear propensity for self-assembly. TEM analysis revealed that these peptides self-assemble into nanoparticles or nanofibers. FT-IR and UV-Vis spectroscopy confirmed the formation of ordered β-sheet structures in the self-assembled peptides. Further structural characterization of FFVDF by X-ray diffraction and SAED provided insights into the crystal structure and molecular packing arrangement of the nanofibers. Cellular studies demonstrated that the identified peptides were non-toxic to HeLa cells at concentrations up to 200 µM. Several peptides showed efficient cellular uptake, with varying intracellular distributions: some localized to the cell membrane, others distributed within the cytoplasm or lysosomes, and one (LDFPI) observed in the nucleus. Furthermore, the study estimated the frequency of self-assembling pentapeptides within the library, finding that approximately 0.04% of beads displayed a very high fluorescence intensity, suggesting a higher occurrence of self-assembling peptides than initially expected. Investigations into the mechanisms of cellular uptake for certain peptides suggest different endocytic pathways (clathrin-dependent, caveolae-dependent, or micropinocytosis) are involved. Interestingly, some peptides initially formed nanoparticles which transformed into larger nanostructures or nanofibrils over time.

The results demonstrate the effectiveness of the developed fluorescent-activation screening method for rapid and unbiased discovery of self-assembling peptides. The success in identifying multiple peptides with different self-assembly properties and cellular interactions highlights the potential of the OBOC approach for exploring the sequence space of self-assembling peptides. The identified peptides exhibit diverse morphologies and cellular uptake behaviors, suggesting that they could serve as building blocks for constructing nanomaterials with tailored properties for various biomedical applications. The ability to manipulate the self-assembly behavior and cellular interactions of these peptides through changes in amino acid sequence opens up possibilities for designing functional nanomaterials for targeted drug delivery, imaging, and other therapeutic uses. This method also advances our understanding of the factors that govern peptide self-assembly, allowing further exploration of the sequence-structure-function relationships.

This study successfully developed a simple yet powerful method for rapidly discovering self-assembling peptides. Using a fluorescent-activation screening approach based on an OBOC library, the researchers identified eight novel pentapeptides that self-assemble into various nanostructures with different cellular uptake properties. The identified peptides are non-toxic and show potential for various biomedical applications. Future research could explore the use of these peptides in drug or nucleic acid delivery systems and further investigate the structure-activity relationships to optimize their properties for specific applications. The method itself could be expanded to screen for self-assembling peptides with other functionalities or under different environmental conditions.

The study focused on HeLa cells, and the findings may not be directly generalizable to other cell types. The relatively small number of peptides examined may not fully capture the diversity of possible self-assembling sequences. Although the researchers considered possible interference of the FITC label, further studies might quantify the influence of the label on the self-assembly and cellular interactions of the peptides. The estimation of self-assembling peptides is based on fluorescence intensity, requiring further validation through additional analyses. More work is needed to fully understand the mechanisms of action of these peptides in living systems.