Deoxyribozymes (DNAzymes) have demonstrated a significant capacity for biocomputing and hold promise for information processing within advanced biological devices if several key capabilities are developed. One required capability is reusehaving DNAzyme logic gates be cyclically, and controllably, activated and deactivated. We designed an oligonucleotide-based system for DNAzyme reuse that could (1) remove previously bound inputs by addition of complementary oligonucleotides via toe-hold mediated binding and (2) diminish output signal through the addition of quencher-labeled oligonucleotides complementary to the fluorophore-bound substrate. Our system demonstrated, for the first time, the ability for DNAzymes to have their activity toggled, with activity returning to 90–125% of original activity. This toggling could be performed multiple times with control being exerted over when the toggling occurs, with three clear cycles observed before the variability in activity became too great. Our data also demonstrated that fluorescent output of the DNAzyme activity could be actively removed and regenerated. This reuse system can increase the efficiency of DNAzyme-based logic circuits by reducing the number of redundant oligonucleotides and is critical for future development of reusable biodevices controlled by logical operations.

Increased understanding of the molecular components involved in reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring and retain a continuation of their lineage. In this study, we focus on the Mediterranean snail, Theba pisana, a hermaphroditic land snail that has become a highly invasive pest species within agricultural areas throughout the world. Our analysis of T. pisana CNS tissue has revealed gene transcripts encoding molluscan reproduction-associated proteins including APGWamide, gonadotropin-releasing hormone (GnRH) and an egg-laying hormone (ELH). ELH isoform 1 (ELH1) is known to be a potent reproductive peptide hormone involved in ovulation and egg-laying in some aquatic molluscs. Two other non-CNS ELH isoforms were also present in T. pisana (Tpi-ELH2 and Tpi-ELH3) within the snail dart sac and mucous glands. Bioactivity of a synthetic ELH1 on sexually mature T. pisana was confirmed through bioassay, with snails showing ELH1-induced egg-laying behaviours, including soil burrowing and oviposition. In summary, this study presents a detailed molecular analysis of reproductive neuropeptide genes in a land snail and provides a foundation for understanding ELH function.

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