Nanoplasmonic molecular ruler for nuclease activity and dna footprinting

Abstract

This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplamonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

1 . A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
 a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least two restriction sites positioned such that the conjugates cleaved at each of the restriction sites, provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at the other restriction sites and/or the intact conjugate.   
     
     
         2 . The nanoplasmon resonance ruler of  claim 1 , wherein said nucleic acid is a double-stranded DNA, or a double-stranded RNA. 
     
     
         3 . The nanoplasmon ruler of  claim 2 , wherein said nucleic acid is a double-stranded nucleic acid comprising a first strand attached to said nanoparticle and a second strand hybridized to said first strand, but not otherwise attached to said nanoparticle. 
     
     
         4 . The nanoplasmon resonance ruler of  claim 1 , wherein said nucleic acid comprises at least three different restriction sites. 
     
     
         5 . The nanoplasmon resonance ruler of  claim 1 , wherein said nucleic acid further comprises a protein binding site. 
     
     
         6 . (canceled) 
     
     
         7 . The nanoplasmon resonance ruler of  claim 1 , wherein said nucleic acid ranges in length from 3 nucleotides to about 500 nucleotides. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle is selected from the group consisting of a nanosphere, a nanocrescent, a nanowire, a nanohorn, a nanotube, a nanopyramid, a nanorod, a nanotetrepod, a single- or multi-layered nanodisk, and a nanohorn. 
     
     
         11 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle comprises a metal or semiconductor material. 
     
     
         12 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle comprises a material selected from the group consisting of a noble metal, a noble metal alloy, a noble metal composite. 
     
     
         13 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle comprises a material selected from the group consisting of gold, gold alloy, silver, silver alloy, copper, copper alloy, platinum, platinum alloy, CdSe semiconductor, CdS semiconductor, CdSe coated with ZnS, magnetic colloidal materials, ZnS, ZnO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S3, In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs. 
     
     
         14 . (canceled) 
     
     
         15 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle size ranges from about 5 nm to about 150 nm. 
     
     
         16 . (canceled) 
     
     
         17 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle surface is functionalized for attachment of the nucleic acid. 
     
     
         18 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle surface is functionalized with a phosphine layer. 
     
     
         19 . The nanoplasmon resonance ruler of  claim 1 , wherein the nanoparticle surface is silica coated and functionalized using amino-silane molecules. 
     
     
         20 . The nanoplasmon resonance ruler of  claim 1 , wherein the conjugate comprises from about 10 to about 10,000 nucleic acid molecules per nanoparticle. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The nanoplasmon resonance ruler of  claim 1 , wherein the conjugate is immobilized on a substrate appropriate for surface plasmon resonance. 
     
     
         25 . The nanoplasmon resonance ruler of  claim 1 , wherein the conjugate is electrostatically immobilized on a glass, quartz, or ceramic surface. 
     
     
         26 . A substrate for identifying binding sites of molecules that bind to nucleic acids, said substrate comprising:
 a substrate appropriate for surface plasmon resonance measurements bearing a collection of nanoplasmon resonance rulers attached thereto, each ruler comprising a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, whereby changes in the size of the nucleic acid are detectable through changes in the plasmon resonance signature of the conjugate, and where a plurality of members of said collection comprise different nanoconjugate species and said members are addressed.   
     
     
         27 . The substrate of  claim 26 , wherein the conjugates are spatially addressed. 
     
     
         28 . The substrate of  claim 26 , wherein the substrate bears at least three different nanoconjugate species. 
     
     
         29 .- 36 . (canceled) 
     
     
         37 . The substrate of  claim 26 , wherein the nanoparticles are selected from the group consisting of a nanosphere, a nanocrescent, a nanowire, a nanohorn, a nanotube, a nanopyramid, a nanorod, a nanotetrepod, a single- or multi-layered nanodisk, and a nanohorn. 
     
     
         38 - 49 . (canceled) 
     
     
         50 . The substrate of  claim 26 , wherein the nucleic acid comprises a dsDNA oligonucleotide of SEQ ID NO:15, wherein X in SEQ ID NO: 15 is any inserted DNA sequence or feature and ranges in length from about 3 to about 50 nucleotides. 
     
     
         51 . The nanoplasmon resonance ruler of  claim 50 , wherein X comprises a sequence selected from the group consisting of: a transcription factor binding site, a mismatch sequence, a single nucleotide polymorphism, an epigenetically changed sequence, a protein binding site, and an enzyme binding site. 
     
     
         52 . A nanoplasmon resonance ruler, said ruler comprising:
 a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, whereby changes in the size of nucleic acid are detectable through changes in the plasmon resonance signature of the conjugate.   
     
     
         53 . (canceled) 
     
     
         54 . The nanoplasmon ruler of  claim 52 , wherein said nucleic acid is a double-stranded comprising a first strand attached to said nanoparticle and a second strand hybridized to said first strand, but not otherwise attached to said nanoparticle. 
     
     
         55 .- 61 . (canceled) 
     
     
         62 . The nanoplasmon resonance ruler of  claim 52 , wherein the nanoparticle is selected from the group consisting of nanosphere, a nanocrescent, a nanowire, a nanohorn, a nanotube, a nanopyramid, a nanorod, a nanotetrepod, a single- or multi-layered nanodisk, and a nanohorn. 
     
     
         63 - 68 . (canceled) 
     
     
         69 . A method of identifying the presence of and/or determining the location of a binding site of a molecule that binds to a nucleic acid, said method comprising:
 providing a conjugate comprising a nanoparticle attached to a nucleic acid comprising the binding site for said molecule;   contacting said conjugate ruler with a composition expected to contain said molecule under conditions where said molecule binds to said nucleic acid to form a bound conjugate;   digesting said nucleic acid with a non-specific exonuclease and detecting the change in nanoplasmon resonance resulting from said digestion, where the change in nanoplasmon resonance provides an indicator of the presence of said molecule and the location of the binding site of said molecule to said nucleic acid.   
     
     
         70 - 72 . (canceled) 
     
     
         73 . A method of identifying the presence of and/or characterizing the binding site of a molecule, said method comprising:
 providing a collection of nanoplasmon resonance rulers wherein each ruler comprises a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, whereby changes in the size the nucleic acid are detectable through changes in the plasmon resonance signature of the conjugate, and where a plurality of members of said collection comprise different nucleic acids and said members are addressed;   contacting the collection with said molecule under conditions where said molecule binds to one or more of the nucleic acids comprising the conjugate members of said collection to form a bound conjugate;   digesting nucleic acids with a non-specific exonuclease and detecting the change in nanoplasmon resonance resulting from said digestion, where the change in nanoplasmon resonance provides an indicator of the nanoconjugates in the collection bound by the molecule, and the location of the binding site of said molecule on the nucleic acid(s).   
     
     
         74 . The method of  claim 73 , wherein the nanoconjugates are attached to a substrate compatible with plasmon resonance detection. 
     
     
         75 . The method of  claim 74 , wherein the conjugates are spatially addressed. 
     
     
         76 - 80 . (canceled) 
     
     
         81 . A method of detecting a mismatch in a nucleic acid hybridization, said method comprising:
 providing a conjugate comprising a nanoparticle attached to a single-stranded nucleic acid probe;   contacting said nucleic acid probe with test nucleic acid under conditions where said test nucleic acid can hybridize to said probe nucleic acid despite the presence of a mismatch forming a double stranded nucleic acid;   contacting said nucleic acid probe with a molecule that binds to said mismatch;   digesting the double-stranded nucleic acid with a non-specific exonuclease and detecting the change in nanoplasmon resonance resulting from said digestion, where the change in nanoplasmon resonance provides an indicator of the presence and location of the mismatch.   
     
     
         82 . The method of  claim 81 , wherein said molecule that binds the mismatch is selected from the group consisting of a protein that binds a single base mismatch, a protein that binds a bubble, a protein that binds a loop, a protein that binds a nick, a protein that binds a ssDNA-dsDNA transition, a protein that binds a flap, and a protein that binds a Y-fork. 
     
     
         83 - 90 . (canceled) 
     
     
         91 . A method of detecting a mismatch in a nucleic acid hybridization, said method comprising:
 providing a conjugate comprising a nanoparticle attached to a single-stranded nucleic acid probe;   contacting said nucleic acid probe with test nucleic acid under conditions where said test nucleic acid can hybridize to said probe nucleic acid despite the presence of a mismatch and where the mismatch results in portion of the strands directed away from the nanoparticle remaining single stranded; and   digesting the terminal single-stranded nucleic acid(s) with a single-strand specific exonuclease and detecting the change in nanoplasmon resonance resulting from said digestion, where the change in nanoplasmon resonance provides an indicator of the presence and location of the mismatch.   
     
     
         92 - 104 . (canceled) 
     
     
         105 . A method for detecting a target feature or quantifying a binding event using a nanoplasmon resonance ruler comprising:
 providing a Au-DNA nanoparticle conjugate comprised of a nanoparticle having a ssDNA oligonucleotide tethered thereto, wherein the oligonucleotide contains a restriction site;   hybridizing a complementary oligonucleotide to said ssDNA oligonucleotide, wherein the complementary oligonucleotide contains a target feature or sequence to be probed is inserted at said restriction site;   allowing a protein to bind to said target feature or sequence;   performing DNA digestions using an exonuclease on the nanoconjugate;   observing time-lapse scattering spectra using surface plasmon resonance, wherein the plasmon resonance wavelength of the Au-DNA nanoparticle conjugate is measured as a function of time in the exonuclease reactions, whereby the size of the nanoconjugate and the binding event is detectable through the time-resolved measurement of the nanoplasmon resonance spectra.