Forced Intercalation Peptide Nucleic Acid Probes for the Detection of an Adenosine-to-Inosine Modification.

The deamination of adenosine to inosine is an important modification in nucleic acids that functionally recodes the identity of the nucleobase to a guanosine. Current methods to analyze and detect this single nucleotide change, such as sequencing and PCR, typically require time-consuming or costly procedures. Alternatively, fluorescent "turn-on" probes that result in signal enhancement in the presence of target are useful tools for real-time detection and monitoring of nucleic acid modification. Here we describe forced-intercalation PNA (FIT-PNA) probes that are designed to bind to inosine-containing nucleic acids and use thiazole orange (TO), 4-dimethylamino-naphthalimide (4DMN), and malachite green (MG) fluorogenic dyes to detect A-to-I editing events. We show that incorporation of the dye as a surrogate base negatively affects the duplex stability but does not abolish binding to targets. We then determined that the identity of the adjacent nucleobase and temperature affect the overall signal and fluorescence enhancement in the presence of inosine, achieving an 11-fold increase, with a limit of detection (LOD) of 30 pM. We determine that TO and 4DMN probes are viable candidates to enable selective inosine detection for biological applications.

Systematically Modulating Aptamer Affinity and Specificity by Guanosine-to-Inosine Substitution.

Aptamers are widely used in small molecule detection applications due to their specificity, stability, and cost effectiveness. One key challenge in utilizing aptamers in sensors is matching the binding affinity of the aptamer to the desired concentration range for analyte detection. The most common methods for modulating affinity have inherent limitations, such as the likelihood of drastic changes in aptamer folding. Here, we propose that substituting guanosine for inosine at specific locations in the aptamer sequence provides a less perturbative approach to modulating affinity. Inosine is a naturally occurring nucleotide that results from hydrolytic deamination of adenosine, and like guanine, it base pairs with cytosine. Using the well-studied cocaine binding aptamer, we systematically replaced guanosine with inosine and were able to generate sequences having a range of binding affinities from 230 nM to 80 µM. Interestingly, we found that these substitutions could also modulate the specificity of the aptamers, leading to a range of binding affinities for structurally related analytes. Analysis of folding stability via melting temperature shows that, as expected, aptamer structure is impacted by guanosine-to-inosine substitutions. The ability to tune binding affinity and specificity through guanosine-to-inosine substitution provides a convenient and reliable approach for rapidly generating aptamers for diverse biosensing applications.