Rapid Identification of Microorganisms

Abstract

Methods of labeling, identifying and differentiating microorganisms using functionalized Buckyballs are provided herein. The invention further provides methods for imaging or inhibiting gene expression using functionalized Buckyballs of the invention. The invention also provides a system for labeling, identifying and differentiating microorganisms.

1 - 31 . (canceled) 
     
     
         32 . A method of inhibiting gene expression in a living microorganism, the method comprising:
 a) functionalizing a Buckminsterfullerene molecule with one or more RNA oligonucleotides complementary to one or more mRNA segments of interest corresponding to a gene of interest;   b) hybridizing the one or more RNA oligonucleotides to one or more complementary protecting layers comprising segments of DNA or RNA and optionally a detectable label; and   c) contacting the sample with the functionalized Buckminsterfullerene molecule for a period of time;   wherein the one or more RNA oligonucleotides hybridize with free mRNA in the cytoplasm, preventing transcription and gene expression; and   wherein, the detectable label, if present, is not detected when the protecting layers are hybridized to the one or more RNA oligonucleotides and the detectable label is detected when the protecting layers are not hybridized.   
     
     
         33 . The method of  claim 32 , wherein the one or more RNA oligonucleotides are selected by bioinformatics analysis. 
     
     
         34 . The method of  claim 32 , wherein the protecting layers are about 75% complementary to the corresponding RNA oligonucleotides. 
     
     
         35 . The method of  claim 32 , wherein the one or more RNA oligonucleotides are each independently about 80% to a 100% complementary to the corresponding species specific signature RNA sequences. 
     
     
         36 . The method of  claim 32 , wherein the one or more RNA oligonucleotides each independently comprise about 20 to about 50 individual nucleotides. 
     
     
         37 . The method of  claim 32 , wherein the one or more RNA oligonucleotides are siRNA oligonucleotides. 
     
     
         38 . The method of  claim 32 , wherein the Buckminsterfullerene molecule is selected from the group consisting of C60 Buckminsterfullerene, C70 Buckminsterfullerene and C60-pyrrolidine tris acid Buckminsterfullerene. 
     
     
         39 . The method of  claim 32 , wherein the detectable label is from the group consisting of a fluorescent tag, a radioactive isotope, an amino acid, a nucleic acid, and a peptide. 
     
     
         40 . The method of  claim 39 , wherein the detectable label is selected from the group consisting of glycine, tryptophan, arginine, cysteine, fBSA,  14 C,  125 I, and cy3/6-FAM. 
     
     
         41 . The method of  claim 32 , wherein the detectable label is detected using a method selected from the group consisting of autoradiography, fluorescence microscopy, X-ray fluorescence microscopy, UV-vis spectroscopy, TEM and fluorescent spectroscopy. 
     
     
         42 . The method of  claim 32 , wherein the method does not require sample fixation. 
     
     
         43 . The method of  claim 32 , wherein the microorganism is selected from the group consisting of bacteria, fungi, archaea and protists. 
     
     
         44 . The method of  claim 32 , wherein the microorganism internalizes the functionalized Buckminsterfullerene. 
     
     
         45 . The method of  claim 32 , wherein biological processes can be monitored and profiled by dynamic visualization of mRNA expression. 
     
     
         46 - 73 . (canceled)