In Silico-Ex Vitro Iteration Strategy for Affinity Maturation of Anti-Ricin Peptides and the SPR Biosensing Application

Abstract

The highly toxic plant toxin ricin is one of the most known threatening toxins. Accurate and sensitive biosensing methods for the first emergency response and intoxication treatment, are always pursued in the biodefense field. Screening affinity molecules is the fundamental mainstream approach for developing biosensing methods. Compared with common affinity molecules such as antibodies and oligonucleotide aptamers, peptides have great potential as biosensing modules with more accessible chemical synthesis capability and better batch-to-batch stability than antibodies, more abundant interaction sites, and robust sensing performance towards complex environments. However, anti-ricin peptides are so scant to be screened and discovered, and an advanced screening strategy is the utmost to tackle this issue. Here, we present a new in silico-in vitro iteration-assisted affinity maturation strategy of anti-ricin peptides. We first obtained affinity peptides targeting ricin through phage display with five panning rounds of "coating-elution-amplification-enrichment" procedures. The binding affinity and kinetic parameters characterized by surface plasmon resonance (SPR) showed that we had obtained four peptides owning dissociation constants (K_D) around 2~35 μM, in which peptide PD-2-R5 has the lower K_D of 4.7 μM and higher stable posture to interact with ricin. We then constructed a new strategy for affinity maturity, composing two rounds of in silico-in vitro iterations. Firstly, towards the single-site alanine scanning mutation peptide library, the molecular docking predictions match the SPR evaluation results well, laying a solid foundation for designing a full saturation mutated peptide library. Secondly, plenty of in silico saturation mutation prediction results guided the discovery of peptides PD2-R5-T3 and PD-2-R5-T4 with higher affinity from only a limited number of SPR evaluation experiments. Both evolved peptides had increased affinity by about 5~20 times, i.e., K_D of 230 nM and 900 nM. A primary cellular toxicity assay indicated that both peptides could protect cells against ricin damage. We further established an SPR assay based on PD-2-R5-T3 and PD-2-R5-T4 elongated with an antifouling peptide linkage and achieved good linearity with a sensitivity of 1 nM and 0.5 nM, respectively. We hope this new affinity-mature strategy will find its favorable position in relevant peptide evolution, biosensing, and medical countermeasures for biotoxins to protect society's security and human life better.

Keywords: affinity maturation; biosensing; molecular docking; peptide; phage display; ricin; surface plasmon resonance.

Scheme 1

Schematic of workflow for screening peptides by phage display.

Figure 1

The OD_450nm value of an enriched peptide library against ricin or RCA120 in each round.

Figure 2

The binding OD_450nm of monoclonal phages evolved from the fifth pool for ricin (A) and RCA120 (B) by ELISA. The dotted line was a positive boundary line.

Figure 3

The multi-cycle kinetics (MCK) of peptides towards ricin or RCA120 using SPR.

Figure 4

Molecular docking of peptides with ricin (A), in which yellow: ricin A chain; grey: ricin B chain; red: PD-2-R5; cyan: PD-2-R15; green: PD-2-R19; and pink: PD-2-R23; the interaction sites (B) in which pink: PD-2-R5; forest green: primary active pocket of ricin, and split pea green: second active pocket of ricin; and the heat map of site interactions between four peptides and ricin (C).

Figure 5

The multi-cycle kinetics of saturation-mutated peptides PD-2-R5-T3 and PD-2-R5-T4 and ricin using SPR.

Figure 6

The neutralizing effect of PD-2-R5 and PD-2-R5-C10 peptides (A), PD-2-R5-T3 and PD-2-R5-T3-C10 peptides (B), PD-2-R5-T4 and PD-2-R5-T4-C10 peptides (C) on ricin, and the curve of cytotoxicity with ricin only ((D), IC₅₀ = 1.1 ± 0.1 ng/mL, R² = 0.965) by CCK-8 assay.

Figure 7

The SPR response of ricin at a concentration from 1 nM to 500 nM based on Bio-PPPP-EKEKEKE-PD-2-R5-T3 (A) and Bio-PPPP-EKEKEKE-PD-2-R5-T4 (C). The titration and linear curves (inset) of SPR quantification on Bio-PPPP-EKEKEKE-PD-2-R5-T3 (B) and Bio-PPPP-EKEKEKE-PD-2-R5-T4 (D).

Figure 8

The selectivity of this SPR assay based on Bio-PPPP-EKEKEKE-PD-2-R5-T3 and Bio-PPPP-EKEKEKE-PD-2-R5-T4.