Highly stable and specific molecular beacons encapsulated in cationic lipoplex nanoparticles and application thereof
The present invention discloses a method of detecting the presence of mutated genes, mRNAs or microRNAs in a subject. The method comprises the following steps: (1) Provide a body fluid sample containing cells, circulating tumor cells (CTCs), and/or extracellular vesicles (EVs); and use an analyzer having overhang molecular beacons to measure fluorescence signals generated by interactions between the body fluid sample and the overhang molecular beacons, so as to detect the presence of the mutated genes, mRNAs or microRNA. Furthermore, a biochip comprising a gold coating substrate and tethered lipoplex nanoparticles encapsulating the overhang molecular beacons is also provided in the invention.
1. A nucleic acid hybridization probe for detecting a target nucleic acid, wherein the nucleic acid hybridization probe comprises:
(a) a stem comprising at least 5 base-paired nucleotides, wherein the stem has a first end and a second end;
(b) a loop having at least 3 nucleotides; and
(c) an overhang section comprising at least 3 nucleotides and attached to the first end of the stem,
wherein the overhang section hybridizes to the target nucleic acid;
wherein a first label comprising a fluorophore or chromophore is attached to a first nucleotide within the stem and a second label is attached to a nucleotide that is within the stem and that is complementary base-paired with the first nucleotide, wherein the first nucleotide is situated between two other nucleotides, and wherein a single-point mutated base in the target nucleic acid is complementary to the first nucleotide or either of the two other nucleotides.
2. The nucleic acid hybridization probe of claim 1 , wherein the nucleic acid probe comprises a nucleic acid selected from the group consisting of SEQ ID No 2, SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 9, SEQ ID No 11, SEQ ID No 12 SEQ ID No 13 and their combinations.
3. The nucleic acid hybridization probe of claim 1 , wherein the target nucleic acid is a wild-type or mutant KRAS nucleic acid.
4. The nucleic acid hybridization probe of claim 1 , wherein the second label comprises a quencher.
5. The nucleic acid hybridization probe of claim 1 , wherein the fluorophore is selected from the group consisting of FAM, Cy3, Cy5, iFluorT, iCy3, and iCy5.
6. The nucleic acid hybridization probe of claim 4 , wherein the quencher is selected from the group consisting of BHQ-1 and BHQ-2.
7. The nucleic acid hybridization probe of claim 1 , wherein the first nucleotide within the stem is attached to the overhang section.
8. The nucleic acid hybridization probe of claim 1 , wherein the first nucleotide within the stem base pairs with the single-point mutated base in the target nucleic acid.
9. The nucleic acid hybridization probe of claim 1 , wherein the nucleic acid hybridization probe is encapsulated within a lipoplex nanoparticle.
10. The nucleic acid hybridization probe of claim 9 , wherein the lipoplex nanoparticle is tethered on a substrate.
11. The nucleic acid hybridization probe of claim 10 , wherein the substrate is a gold coating substrate.
12. The nucleic acid hybridization probe of claim 1 , wherein one of the two other nucleotides base pairs with the single-point mutated base in the target nucleic acid.
13. The nucleic acid hybridization probe of claim 3 , wherein the mutant KRAS nucleic acid is selected from the group consisting of KRAS G12C , KRAS G12D , and KRAS G12V .