Full liberation of 2-Aminopurine with nucleases digestion for highly sensitive biosensing

2-Aminopurine (2-AP), a fluorescent isomer of adenine, is a popular fluorescent tag for DNA-based biosensors. The fluorescence of 2-AP is highly dependent on its microenvironment, i.e., almost non-fluorescent and merely fluorescent in dsDNA and ssDNA, respectively, but can be greatly brightened as mononucleotide. In most 2-AP-based biosensors, DNA transformation from dsDNA to ssDNA was employed, while selective digestion of 2-AP-labeled DNA with nucleases represents an appealing approach for improving the biosensor sensitivity. However, some detailed fundamental information, such as the reason for nuclease digestion, the influence of the labeling site, neighboring bases, or the label number of 2-AP for final signal output, are still largely unknown, which greatly limits the utility of 2-AP-based biosensors. In this work, using both steady- and excited-state fluorescence (lifetime), we demonstrated that nuclease digestion resulted in almost full liberation of 2-AP mononucleotides, and was free from labeling site and neighboring bases. Furthermore, we also found that nuclease digestion could lead to multiplexed sensitivity from increasing number of 2-AP labelling, but was not achievable for the conventional biosensors without full liberation of 2-AP. Considering the popularity of 2-AP in biosensing and other related applications, the above obtained information in sensitivity boosting is fundamentally important for future design of 2-AP-based biosensors.