Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Diagnostics

Background:

• Multiplexed nucleic acid sensing is crucial for diagnostics and infectious disease control, especially post-pandemic.
• Nanopore sensing offers highly sensitive, single-molecule detection capabilities.
• Existing methods require improvement in specificity and multiplexing for comprehensive analysis.

Purpose of the Study:

• To develop a nanopore sensor utilizing DNA dumbbell nanoswitches for multiplexed nucleic acid detection.
• To achieve highly specific identification of nucleic acid sequences and bacterial species.
• To demonstrate the sensor's capability for simultaneous detection of multiple targets.

Main Methods:

• Fabrication of DNA dumbbell nanoswitches designed to change topology upon target hybridization.
• Integration of four distinct nanoswitches on a single carrier for multiplexed detection.
• Utilizing nanopore current traces to identify topological changes indicative of target binding.
• Employing barcoded DNA carriers for enhanced specificity and bacterial identification via 16S ribosomal RNA (rRNA) fragments.

Main Results:

• Simultaneous detection of four different nucleic acid sequences was successfully achieved.
• High specificity was demonstrated by distinguishing single base variants in DNA and RNA targets.
• Bacterial species identification, even with high sequence similarity, was accomplished using strain-specific 16S rRNA fragments.
• The DNA dumbbell nanoswitch exhibited a distinct current trace peak upon target binding.

Conclusions:

• DNA dumbbell nanoswitches provide a robust platform for highly specific, multiplexed nanopore-based nucleic acid sensing.
• This technology holds significant potential for advancing clinical diagnostics and infectious disease surveillance.
• The method allows for sensitive and accurate identification of bacterial species at the strain level.