Methods for isothermal molecular amplification with nanoparticle-based reactions

Abstract

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 an Input-Oligonucleotide, the method comprising
 a.) providing a plurality of Particle-A, wherein Particle-A is functionalized with a plurality of Oligonucleotide-1 and a plurality of Oligonucleotide-2,   wherein Oligonucleotide 1 and Oligonucleotide 2 are hybridized to form Duplex 1-2, and have attached oligonucleotide origami;   b.) providing a plurality of Particle-B, wherein Particle-B is functionalized with a plurality of Oligonucleotide-3 and a plurality of Stored-Input-Oligonucleotide,   wherein Oligonucleotide-3 and Stored-Input-Oligonucleotide are hybridized to form Duplex 3-Y, and wherein at least a portion of the sequences of the Input-Oligonucleotide and the Stored-Input-Oligonucleotide are identical; and   c.) mixing the Input-Oligonucleotide, Particle-A and Particle-B, in any order, wherein a cascade reaction occurs so that:   i) the Input-Oligonucleotide fully hybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2 and oligonucleotide origami from Particle-A, and exposing Oligonucleotide-1,   ii) exposed Oligonucleotide-1 hybridizes with Oligonucleotide-3 to form Duplex 1-3, thereby releasing Stored-Input-Oligonucleotide, and   iii) where (i) reoccurs with the Stored-Input-Oligonucleotide replacing the Input-Oligonucleotide until the cascade reaction terminates,   
       wherein the Input-Oligonucleotide is amplified by the release of the Stored-Input-Oligonucleotide. 
     
     
         2 . The method of  claim 1 , wherein the Tm of Duplex Y-2 is at least about 20% greater than the Tm of Duplex 1-2. 
     
     
         3 . The method of  claim 1 , wherein the Tm of Duplex 1-3 is at least about 20% greater than the Tm of Duplex 3-Y. 
     
     
         4 . The method of  claim 1 , wherein the cascade reaction terminates when all of the Stored-Input-Oligonucleotide is released. 
     
     
         5 . The method of  claim 1 , wherein the Input-Oligonucleotide is DNA. 
     
     
         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 and/or about 25 to about 400 Oligonucleotide-3 attached per about 5 nm to about 40 nm diameter of Particle-B. 
     
     
         10 . The method of  claim 1 , wherein there are about 200 Oligonucleotide-1 attached per about 20 nm diameter of Particle-A and/or about 200 Oligonucleotide-3 attached per about 20 nm diameter of Particle-B. 
     
     
         11 . The method of  claim 1 , wherein Oligonucleotide-1 is grafted to Particle-A, and Oligonucleotide-3 is grafted to Particle-B. 
     
     
         12 . The method of  claim 1 , wherein the oligonucleotide origami is rectangular and wherein there are about 2 to about 10 origami per about 5 nm to about 40 nm diameter of Particle-A. 
     
     
         13 . The method of  claim 12 , wherein there are about 3 to about 4 origami per about 20 nm diameter of Particle-A. 
     
     
         14 . The method of  claim 1 , wherein the Input-Oligonucleotide is amplified by a factor of about 50 to about 1000. 
     
     
         15 . The method of  claim 1 , wherein the cascade reaction is under isothermal conditions. 
     
     
         16 . The method of  claim 1 , wherein the relative amount of Particle-A:Particle-B is about 1:1. 
     
     
         17 . A method for identifying an Analyte-Oligonucleotide, the method comprising
 a.) providing a plurality of Particle-A, wherein Particle-A is functionalized with a plurality of Oligonucleotide-1 and a plurality of Oligonucleotide-2,   wherein Oligonucleotide-1 and Oligonucleotide-2 are hybridized to form Duplex 1-2, and have attached oligonucleotide origami;   b.) providing a plurality of Particle-B, wherein Particle-B is functionalized with a plurality of Oligonucleotide-3 and a plurality of Target-Oligonucleotide,   wherein Oligonucleotide-3 and Target-Oligonucleotide are hybridized to form Duplex 3-Y; and   c.) mixing the Analyte-Oligonucleotide, Particle-A and Particle-B, in any order, wherein if the sequence of the Analyte-Oligonucleotide and the sequence of the Target-Oligonucleotide are identical, a cascade reaction occurs so that:   i) the Analyte-Oligonucleotide hybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2 and oligonucleotide origami from Particle-A, and exposing Oligonucleotide-1,   ii) exposed Oligonucleotide-1 partially hybridizes with Oligonucleotide-3 to form Duplex 1-3, thereby releasing Target-Oligonucleotide, and   iii) where (i) reoccurs with the Target Oligonucleotide replacing the Analyte-Oligonucleotide,   
       wherein the Analyte-Oligonucleotide is identified as the Target-Oligonucleotide by aggregation of Particle-A and Particle-B. 
     
     
         18 . The method of  claim 17 , wherein the Tm of Duplex Y-2 is at least about 20% greater than the Tm of Duplex 1-2. 
     
     
         19 . The method of  claim 17 , wherein the Tm of Duplex 1-3 is at least about 20% greater than the Tm of Duplex 3-Y. 
     
     
         20 . A sensor apparatus for identifying an Analyte-Oligonucleotide, the apparatus comprising
 a.) a plurality of Particle-A, wherein Particle-A is functionalized with a plurality of Oligonucleotide-1 and a plurality of Oligonucleotide-2,   wherein Oligonucleotide-1 and Oligonucleotide-2 are hybridized to form Duplex 1-2, and have attached oligonucleotide origami; and   b.) a plurality of Particle-B, wherein Particle-B is functionalized with a plurality of Oligonucleotide-3 and a plurality of Target-Oligonucleotide,   wherein Oligonucleotide-3 and Target-Oligonucleotide are hybridized to form Duplex 3-Y,   wherein the Analyte-Oligonucleotide is mixed with Particle-A and Particle-B, in any order, wherein if at least a portion of the Analyte-Oligonucleotide is identical to the Target-Oligonucleotide, a cascade reaction occurs so that:   i) the Analyte-Oligonucleotide fully hybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2 and oligonucleotide origami from Particle-A, and exposing Oligonucleotide-1,   ii) exposed Oligonucleotide-1 partially hybridizes with Oligonucleotide-3 to form Duplex 1-3, thereby releasing Target-Oligonucleotide, and   iii) where (i) reoccurs with the Target-Oligonucleotide replacing the Analyte-Oligonucleotide,   
       wherein the Analyte-Oligonucleotide is identified as the Target-Oligonucleotide if there is an aggregation of Particle-A and Particle-B.