Single molecule nucleic acid nanoparticles

Abstract

The present technology relates to a nanoparticle platform based on the unique and varied properties of DNA. Circular DNA can be replicated using a strand displacing polymerase to generate long linear concatamers of controllable length that spontaneously fold into a ball conformation due to internal base-pairing. These balls of DNA are discreet particles that can be made in variable sizes on a nanometer size scale in a scalable manner. The particles can be used in a variety of manners, discussed herein, including specific targeting, drug delivery to cancer cells, and diagnostics. Nanoparticles may also serve as multifunctional platforms for the integration of many currently used cancer therapeutic techniques.

1 .- 61 . (canceled) 
     
     
         62 . A method of making a nanoparticle comprising:
 contacting a circular single-stranded nucleic acid template with a nucleic acid polymerase, wherein said template includes a module sequence which binds to a target molecule, wherein the module sequence in monovalent form has low affinity for the target molecule and in multivalent form binds to the target molecule with a high avidity; and   amplifying said template with said polymerase to produce said nanoparticle, wherein said nanoparticle comprises a continuous strand of nucleic acid comprising a concatamer of said module sequence.   
     
     
         63 . The method of  claim 62 , wherein the target molecule is bound to a surface. 
     
     
         64 . The method of  claim 62 , wherein said nucleic acid template is DNA. 
     
     
         65 . The method of  claim 62 , wherein said nucleic acid polymerase is a strand displacing polymerase. 
     
     
         66 . The method of  claim 62 , wherein said amplifying has a duration of more than 1 minute. 
     
     
         67 . The method of  claim 62 , further comprising circularizing a linear nucleic acid template to produce said circular nucleic acid template. 
     
     
         68 . A non-naturally occurring nanoparticle made according to the method of  claim 62 . 
     
     
         69 . The nanoparticle of  claim 68 , wherein said nucleic acid comprises DNA, wherein said DNA is more than 100 kb in length. 
     
     
         70 . The nanoparticle of  claim 68 , wherein said DNA comprises a sequence encoding a sequence selected from a siRNA, reporter gene, therapeutic protein, and CpG sequence. 
     
     
         71 . The nanoparticle of  claim 68 , further comprising a nucleic acid intercalating drug. 
     
     
         72 . The nanoparticle of  claim 68 , further comprising an oligonucleotide-linked entity selected from the group consisting of an aptamer, drug, peptide, and siRNA. 
     
     
         73 . A liposome comprising the nanoparticle of  claim 68 . 
     
     
         74 . A pharmaceutical composition comprising the nanoparticle of  claim 68 . 
     
     
         75 . A method of treating cancer comprising administering the pharmaceutical composition of  claim 74  to a subject in need thereof. 
     
     
         76 . A method for identifying nanoparticles comprising a module sequence capable of binding to a target molecule with high avidity comprising:
 generating a library of nanoparticles comprising putative module sequences using the method of  claim 62 ;   contacting said library to a target molecule; and   selecting for a nanoparticle that binds said target molecule, wherein the module sequence in monovalent form has low affinity for the target molecule and in multivalent form binds to the target molecule with a high avidity.   
     
     
         77 . A non-naturally occurring nanoparticle comprising a single-strand nucleic acid comprising a continuous strand of nucleic acid comprising a concatameric module sequence which binds to a target molecule, wherein the module sequence in monovalent form has low affinity for the target molecule and in multivalent form binds to the target molecule with a high avidity. 
     
     
         78 . The nanoparticle of  claim 77 , wherein the target molecule is bound on the surface. 
     
     
         79 . The nanoparticle of  claim 77 , wherein said nucleic acid comprises DNA, wherein said DNA is more than 100 kb in length. 
     
     
         80 . The nanoparticle of  claim 79 , wherein said DNA comprises a sequence encoding a sequence selected from a siRNA, reporter gene, therapeutic protein, and CpG sequence. 
     
     
         81 . The nanoparticle of  claim 77 , further comprising a nucleic acid intercalating drug. 
     
     
         82 . The nanoparticle of  claim 77 , further comprising an oligonucleotide-linked entity selected from the group consisting of an aptamer, drug, peptide, and siRNA. 
     
     
         83 . A liposome comprising the nanoparticle of  claim 77 . 
     
     
         84 . A pharmaceutical composition comprising the nanoparticle of  claim 77   
     
     
         85 . A method of treating cancer comprising administering the pharmaceutical composition of  claim 84  to a subject in need thereof. 
     
     
         86 . A method of identifying a target comprising:
 contacting said target with the nanoparticle of  claim 77 , wherein said module sequence selectively binds to said target; and   identifying binding of said module sequence to said target.   
     
     
         87 . A library of nanoparticles comprising at least two populations of nanoparticles, wherein said each of said at least two populations comprise nanoparticles comprising a single-strand nucleic acid comprising a continuous strand of nucleic acid comprising a concatamer of at least one different module sequence which binds to a target molecule bound, wherein the module sequence in monovalent form has low affinity for the target molecule and in multivalent form binds to the target molecule with a high avidity.