Methods for isothermal molecular amplification with nanoparticle-based reactions
The present method of detection involves increasing an amount of analyte molecules by an isothermal molecular amplification approach. In the present approach a starting molecule of interest may be amplified through a reaction it induces with specifically engineered and functionalized particles, namely protected particles A and storage particles B. This reaction may result in a set of output DNA molecules that is larger in number than the input DNA molecules. Thus the reaction between nanoparticles for amplification of a certain DNA sequence (input DNA molecules) may occur when there is a match with a targeted molecule (stored molecules on storage particles B) and if the DNA sequence of the input DNA molecules does not match (partially or completely) the targeted molecule the reaction may not occur. Without a certain molecular input of the input DNA molecule the reaction may not occur.
1. A method for amplifying a molecular signal of an Input-Oligonucleotide, the method comprising
a) providing a plurality of Particle-A protected by oligonucleotide origami, wherein Particle-A is functionalized with a plurality of Oligonucleotide-1, wherein a sequence in Oligonucleotide 1 complementary to a sequence in a plurality of Oligonucleotide 2 hybridize to form Duplex 1-2, and sequence in Oligonucleotide 2 complementary to a sequence in oligonucleotide origami hybridize;
b) providing a plurality of Particle-B, wherein Particle-B is functionalized with a plurality of Oligonucleotide-3, wherein a sequence in Oligonucleotide-3 complementary to a sequence in a plurality of Stored-Input-Oligonucleotide hybridize to form Duplex 3-Y; and
c) mixing the Input-Oligonucleotide, plurality of origami protected Particle-A and plurality of Particle-B, in any order, wherein a cascade reaction occurs so that:
i) sequence in the Input-Oligonucleotide complementary to sequence in Oligonucleotide-2 hybridize to form Duplex Y-2, thereby removing Oligonucleotide-2 and the oligonucleotide origami, and exposing Oligonucleotide-1 on Particle A,
ii) sequence in Oligonucleotide-1 complementary to sequence in Oligonucleotide-3 hybridize to form Duplex 1-3, thereby releasing the Stored-Input-Oligonucleotide hybridized to the sequence in Oligonucleotide-3,
iii) sequence in the Stored-Input Oligonucleotide released in ii) complementary to sequence in Oligonucleotide-2 hybridize to form Duplex Y-2, thereby removing Oligonucleotide-2 and the oligonucleotide origami, and exposing Oligonucleotide-1 on Particle A to hybridize to complementary sequence in Oligonucleotide-3 to form Duplex 1-3, thereby releasing the Stored-Input-Oligonucleotide hybridized to the sequence in Oligonucleotide-3, and,
iv) where (iii) reoccurs until the cascade reaction terminates, when Particle A are fully deprotected.
2. The method of claim 1 , wherein the cascade reaction terminates by a first occurrence of either complete deprotection of origami protected Particle A or the release of all Stored-Input-Oligonucleotide from Duplex 3-Y.
3. The method of claim 1 , wherein Duplex Y-2 has a Tm 20% greater than the Tm of Duplex 1-2.
4. The method of claim 1 , wherein Duplex 1-3 has a Tm 20% greater than the Tm of Duplex 3-Y.
5. The method of claim 1 , wherein the Input-Oligonucleotide is DNA or RNA.
6. The method of claim 1 , wherein the Particle-A and the Particle-B are nanoparticles from about 5 to about 100 nm in diameter.
7. The method of claim 6 , wherein the nanoparticles are selected from the group consisting of Au, Ag, Cu, Pt, Pd and combinations thereof.
8. The method of claim 1 , wherein the Input-Oligonucleotide consists of about 5 to about 120 bases.
9. The method of claim 1 , wherein there are about 25 to about 400 Oligonucleotide-1 attached per about 5 nm to about 40 nm diameter of Particle-A.
10. The method of claim 1 , wherein there are about 25 to about 400 Oligonucleotide-3 attached per about 5 nm to about 40 nm diameter of Particle-B.
11. The method of claim 1 , wherein the oligonucleotide origami is 2-dimensional or 3-dimensional.
12. The method of claim 1 , wherein the oligonucleotide origami is rectangular, round or polyhedron.
13. The method of claim 1 , wherein the oligonucleotide origami is rectangular.
14. The method of claim 1 , wherein there are about 3 to about 4 origami per about 20 nm diameter of Particle-A.
15. The method of claim 1 , wherein the cascade reaction is under isothermal conditions.
16. The method of claim 1 , wherein the cascade reaction is under room temperature.
17. The method of claim 1 , wherein the released Stored-Input-Oligonucleotide is measured.
18. The method of claim 17 , wherein the amplified molecular signal of the released Stored-Input-Oligonucleotide is measured.