チクロチドを用いたマイクロ流体プラットフォーム上でのRNA検出

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 trans-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 R,R-THF monomers within the thyclotide backbone promotes a right-handed helix favoring binding to target nucleic acids. Bio-orthogonal thyclotide containing S,S-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.