Bead Bound Combinatorial Oligonucleoside Phosphorothioate And Phosphorodithioate Aptamer Libraries

Abstract

The present invention includes composition and methods for making and using a combinatorial library having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

1 - 30 . (canceled) 
     
     
         30 . A method for aptamer selection comprising the steps of:
 dispersing a one-aptamer, one-bead combinatorial bead library into a two-dimensional matrix;   scanning for aptamer beads that generate a detectable signal from interaction between the one or more aptamer beads and a target; and   picking one or more aptamer beads based on the detectable signal from within the matrix.   
     
     
         31 . The method of  claim 30 , further comprising the step of extracting the target from the aptamer bead. 
     
     
         32 . The method of  claim 30 , further comprising the step of identifying the target by mass spectrometry after liquid chromatography. 
     
     
         33 . The method of  claim 30 , wherein the one-aptamer, one-bead combinatorial bead library is dispered within the matrix by molecular printing. 
     
     
         34 . The method of  claim 30 , wherein the one-aptamer, one-bead combinatorial bead library is dispered within the matrix by molecular printing is via an inkjet printer. 
     
     
         35 . The method of  claim 30 , wherein the matrix comprises a gel, a polymer, a thixotropic agent, a glass or a silicon matrix. 
     
     
         36 . The method of  claim 30 , further comprising the step of separating the target into one or more peptides prior to separation by liquid chromatography. 
     
     
         37 . The method of  claim 30 , wherein the steps of identifying the target by mass spectrometry is preceded by the steps of extracting and separating the proteins by liquid chromatography. 
     
     
         38 . The method of  claim 30 , wherein the steps of identifying the target using mass spectrometry comprises matrix assisted laser desorption ionization (MALDI) mass spectrometry. 
     
     
         39 . The method of  claim 30 , wherein the library comprises an S-ODN library. 
     
     
         40 . The method of  claim 30 , wherein the library comprises an S 2 -ODN library. 
     
     
         41 . The method of  claim 30 , wherein each of the aptamers is further modified to comprise a colorimetric agent. 
     
     
         42 . The method of  claim 30 , wherein each of the aptamers further comprises one or more bases that are attached to a fluorophor. 
     
     
         43 . The method of  claim 30 , wherein each of the aptamers further comprises one or more fluorophors attached to the 5′ end, the 3′ end or internally within the aptamers. 
     
     
         44 . The method of  claim 30 , further comprising the complementary strand to the aptamer. 
     
     
         45 . The method of  claim 30 , wherein the aptamer is defined further as a thioaptamer. 
     
     
         46 . The method of  claim 30 , wherein the aptamer comprises a thioaptamer wherein one or more but less than all of the linkages comprising one or more of the following: rATP(αS), rUTP(αS), rGTP(αS), rCTP(αS), rATP(αS 2 ), rUTP(αS 2 ), rGTP(αS 2 ), rCTP(αS 2 ), rATP(αS), dTTP(αS), dGTP(αS), dCTP(αS), dATP(αS 2 ), dTTP(αS 2 ), dGTP(αS 2 ) and dCTP(αS 2 ). 
     
     
         47 . The method of  claim 30 , wherein the target is labeled with an enzyme, a dye, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore. 
     
     
         48 . The method of  claim 30 , wherein the target is detectable with an enzyme, a radioisotope, an electron dense particle, a magnetic particle, a fluorescent agent, an antibody, a magnetic particle or a chromophore. 
     
     
         49 . The method of  claim 30 , wherein the aptamer bead is further processed to remove the target bound to the aptamer bead. 
     
     
         50 . The method of  claim 30 , wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ. 
     
     
         51 . The method of  claim 30 , aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ by proteolysis and transferred to the inlet of an LC-MS or an LC-MS/MS. 
     
     
         52 . The method of  claim 30 , wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis, wherein the MALDI-MS analysis is selected from the group consisting of MALDI-TOF/MS, MALDI-TOF/TOF-MS and MALDI-Q-TOF-MS. 
     
     
         53 . The method of  claim 30 , wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for LC-MS analysis. 
     
     
         54 . The method of  claim 30 , wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis. 
     
     
         55 . The method of  claim 30 , wherein the aptamer bead is acquired by a scanning robotic head and the target is extracted from the aptamer bead in situ for MALDI-MS analysis by SELDI ionization. 
     
     
         56 . The method of  claim 30 , wherein the aptamer bead is further processed to remove the target bound to the aptamer bead and analyzing the target by MS, MS/MS, MALDI-TOF, MALDI-TOF-MS, direct sequencing. 
     
     
         57 . The method of  claim 56 , wherein the MALDI ionization step is a SELDI ionization. 
     
     
         58 . The method of  claim 30 , wherein the aptamer bead is further processed to remove the target bound to the aptamer bead and analyzing the target by binding a second detectable label to the target. 
     
     
         59 . The method of  claim 30 , wherein the matrix comprises a polyacrylamide gel, an alkyd resin or a silica-lipid. 
     
     
         60 . The method of  claim 30 , wherein picking the beads is selected from picking manually, semi-manually or non-manually. 
     
     
         61 . The method of  claim 30 , wherein the target is selected from peptides, proteins, nucleic acids, carbohydrates, lipids or combinations thereof. 
     
     
         62 - 81 . (canceled)