In Vitro -Selected DNA Aptamers Enable Detection of the Ancestral Peptide 3C8Y in [FeFe]-Hydrogenase CpI and in Metal-Rich, Acidic Environments: Implications for Astrobiology

Selecting molecular biosignatures of unequivocal biological origin and sufficient complexity is essential for developing next-generation biosensors in astrobiology-driven missions. Among the most promising candidates are ancestral peptides, molecular relics that preserve functional signatures of early life. Here, we report the in-vitro selection and characterization of single-stranded DNA aptamers that specifically bind to the 15-amino acid 3C8Y peptide, derived from the [FeFe]-hydrogenase CpI of Clostridium pasteurianum . This ancestral peptide, a candidate biosignature of anaerobic metabolism, contains metal-binding cysteines associated with [FeS] clusters. Three DNA aptamers were selected through a Systematic Evolution of Ligands by EXponential Enrichment-based approach and subsequently validated using colorimetric ELONA assays. The aptamers exhibited high affinity ( K _d in the low nanomolar range) and robust specificity toward both the synthetic 3C8Y peptide (compared with three structurally related peptides) and the heterologously expressed CpI protein in native and denatured states (relative to two other hydrogenases). Dual-aptamer sandwich ELONA confirmed the recognition of distinct binding sites, enabling sensitive detection of the peptide in both free and [FeS]-clustered states. Notably, the aptamers detected 3C8Y-containing targets in complex environmental samples from Río Tinto (Spain), a metal-rich acidic analog, but not in samples from less iron-abundant Icelandic geothermal sites, which are dominated by photosynthetic organisms that do not express CpI hydrogenases. These findings support the use of in-vitro -selected aptamers for label-free detection of oxygen-sensitive metalloproteins in complex matrices, with promising applications in astrobiology.