RNA detection on a microfluidic platform using Thyclotides

Nucleic acid detection is commonly used to diagnose pathogenic and genetic diseases. Diagnostics based on polymerase chain reaction (PCR) are routinely used to amplify and signal the presence of a target nucleic acid, but PCR-based methods are difficult to deploy at the patient's point-of-care and in resource-limited environments. This study reports a novel nucleic acid detection assay that utilizes chemically modified PNAs to directly detect a target RNA without enzymatic amplification. By incorporating <i>trans</i>-3,4-diaminotetrahydrofuran units into a PNA backbone, THF-PNAs (also called thyclotides) were designed with both enhanced binding affinity to target nucleic acids and bio-orthogonal properties that promote thyclotide-to-thyclotide binding over nucleic acid binding. Specifically, incorporation of <i>R,R</i>-THF monomers within the thyclotide backbone promotes a right-handed helix favoring binding to target nucleic acids. Bio-orthogonal thyclotide containing <i>S,S</i>-THF monomers promotes a left-handed helix, preventing their binding to natural nucleic acids. Complementary bio-orthogonal thyclotides can bind to each other in the presence of competing RNA sequences, and these unique properties were used in combination with gold nanoparticles to develop a detection signal. By employing both THF stereochemistries in different thyclotide sequences, a prototype microfluidic assay was developed to detect synthetic HIV-1 RNA and signal its presence using silver-based enhancement of surface-bound gold nanoparticles. The limit of detection for this assay was 0.5 pM of synthetic HIV-1 RNA, which is a significant (~100-fold) improvement over earlier PNA-based detection system. Concentration-dependent variation in detection signal intensity allows for semi-quantitative determination of different RNA concentrations. A scrambled RNA control sequence does not interfere with detection. All results were obtained without using enzymatic amplification. The thyclotides in this study may be used in more advanced diagnostic technologies.