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. Full article

Conotoxins (CTXs) are a variety of mixed polypeptide toxins, among which α-conotoxin MI (CTX-MI) is the most toxic. Serious toxic symptoms, a lack of counteracting drugs, and cumbersome detection processes have made CTX-MI a hidden danger for humans. One of the obstacles to resolving this problem is the absence of specific recognition elements. Aptamers have shown great advantages in the fields of molecule detection, drug development, etc. In this study, we screened and characterized aptamers for CTX-MI through a programmed process. MBMI-01c, the isolated aptamer, showed great affinity, with an affinity constant (K_D) of 0.524 μM, and it formed an antiparallel G-quadruplet (GQ) structure for the specific recognition of CTX-MI. Additionally, an aptasensor based on the biolayer interferometry (BLI) platform was developed and displayed high precision, specificity, and repeatability with a limit of detection (LOD) of 0.26 μM. This aptasensor provides a potential tool for the rapid detection of CTX-MI in 10 min. The aptamer can be further developed for the enrichment, detoxification, and biological studies of CTX-MI. Additionally, the programmed process is applicable to screening and characterizing aptamers for other CTXs. Full article