Methods of enzymatic discrimination enhancement and surface-bound double-stranded dna

Abstract

Methods for discriminating between fully complementary hybrids and those that differ by one or more base pairs and libraries of unimolecular, double-stranded oligonucleotides on a solid support. In one embodiment, the present invention provides methods of using nuclease treatment to improve the quality of hybridization signals on high density oligonucleotide arrays. In another embodiment, the present invention provides methods of using ligation reactions to improve the quality of hybridization signals on high density oligonucleotide arrays. In yet another embodiment, the present invention provides libraries of unimolecular or intermolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.

1 . A method for identifying a base, the method comprising:
 a) hybridizing:
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length, and 
 a target nucleic acid, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; 
   b) hybridizing the target-oligonucleotide probe hybrid complexes with a pool of labeled, ligatable oligonucleotide probes of a preselected length,   c) ligating the labeled ligatable oligonucleotide to the target-oligonucleotide hybrid complex with a ligase; and   d) identifying which base has specifically interacted with the target nucleic acid as an indication of a subsequence that is complementary.   
     
     
         2 . The method according to  claim 1  wherein the target nucleic acid is ribonucleic acid (RNA). 
     
     
         3 . The method according to  claim 1  wherein the target nucleic acid is deoxyribonucleic acid (DNA). 
     
     
         4 . The method of  claim 1 , wherein the array of target; oligonucleotide hybrid complexes has been formed on a substrate comprising beads. 
     
     
         5 . A method for analyzing a target nucleic acid, the method comprising:
 a) providing a reaction system comprising:
 a heating mechanism 
 at least one cavity next to the heating mechanism and 
 a fluorescence detector next to the cavity 
   b) providing a reaction chamber comprising
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length 
 a target nucleic acid 
   c) hybridizing the target nucleic acid to at least one of the oligonucleotides to form target-oligonucleotide hybrid complexes of complementary subsequences of known sequence using the heating mechanism;   d) contacting the target-oligonucleotide probe hybrid complexes with a ligase and a pool of labeled, ligatable oligonucleotide probes of a preselected length,   e) positioning the reaction chamber to the cavity such that substrate surface faces the fluorescence detector; and   f) determining which of the oligonucleotides contain the labeled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of the target nucleic acid.   
     
     
         6 . A diagnostic kit comprising:
 a) a heating mechanism;   b) at least one cavity next to the heating mechanism;   c) a fluorescence detector next to the cavity   d) a reaction chamber;   e) at least one substrate comprising an array of oligonucleotide probes of a preselected length;   f) a target oligonucleotide to be sequenced;   g) a pool of labeled, ligatable oligonucleotide probes of a preselected length; and   h) a ligase, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence.   
     
     
         7 . A data analysis method for analyzing target oligonucleotide sequencing data comprising:
 a) scanning a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex, wherein the labeled oligonucleotide provides an intensity of a signal;   b) processing the intensity of the signal wherein a plurality of subsequences are identified;   c) executing a sorting program to sort all of the subsequences based on a defined hierarchy wherein the defined hierarchy sorts the subsequences in order; and   d) determining which subfragments have been found in the target oligonucleotide sequence.   
     
     
         8 . A data analysis system for analyzing target oligonucleotide sequencing data comprising:
 a) a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex;   b) an automated scanning mechanism wherein the automated scanning mechanism scans the substrate identifying a plurality of subsequences;   c) a sorting program to sort all of the subsequences using a defined hierarchy wherein the defined hierarchy sorts the subsequences and determines, in order, which subfragments have been found in the target sequence.   
     
     
         9 . A computer-readable medium programmed with instructions for analyzing data target oligonucleotide sequencing data comprising computer-executable instructions for causing a programmable processor to execute a method comprising:
 a) receiving a plurality of intensities signals wherein the signal identifies a subsequence from a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex;   b) sorting the subsequences in order using a defined hierarchy wherein the defined hierarchy sorts and determines, in order which subfragments have been found in the target sequence; and   c) retaining the sorted subsequences in the computer-readable medium.   
     
     
         10 . A method for identifying a base, the method comprising:
 a) hybridizing:
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length, and 
 a target nucleic acid, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; 
   b) contacting the target-oligonucleotide hybrid complexes with a nuclease under conditions such that the nuclease preferentially cleaves target-oligonucleotide hybrid complexes; and   c) identifying which base has specifically interacted with the target nucleic acid as an indication of a subsequence that is complementary.   
     
     
         11 . The method according to  claim 10  wherein the target nucleic acid is ribonucleic acid (RNA). 
     
     
         12 . The method according to  claim 10  wherein the nuclease is an RNA nuclease. 
     
     
         13 . The method according to  claim 10  wherein the target nucleic acid is deoxyribonucleic acid (DNA). 
     
     
         14 . The method according to  claim 10  wherein the nuclease is a DNA nuclease. 
     
     
         15 . The method of  claim 10 , wherein the array of target; oligonucleotide hybrid complexes has been formed on a substrate comprising beads. 
     
     
         16 . A method for analyzing a target nucleic acid, the method comprising:
 a) providing a reaction system comprising:
 a heating mechanism 
 at least one cavity next to the heating mechanism and 
 a fluorescence detector next to the cavity 
   b) providing a reaction chamber comprising:
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length 
 a target nucleic acid 
   c) hybridizing the target nucleic acid to at least one of the oligonucleotides to form target-oligonucleotide hybrid complexes of complementary subsequences of known sequence using the heating mechanism;   d) contacting the target-oligonucleotide hybrid complexes with a nuclease under conditions such that the nuclease preferentially cleaves target-oligonucleotide hybrid complexes;   e) positioning the reaction chamber to the cavity such that substrate surface faces the fluorescence detector; and   f) determining which of the oligonucleotides have specifically interacted with subsequences in the target nucleic acid as an indication of a subsequence that is complementary to a subsequence of the target nucleic acid.   
     
     
         17 . A diagnostic kit comprising:
 a) a heating mechanism;   b) at least one cavity next to the heating mechanism;   c) a fluorescence detector next to the cavity and d) a reaction chamber;   d) at least one substrate comprising an array of oligonucleotide probes of a preselected length;   e) a target oligonucleotide to be sequenced;   f) a pool of labeled, ligatable oligonucleotide probes of a preselected length; and   g) a nuclease, wherein the nuclease preferentially cleaves target-oligonucleotide hybrid complexes.   
     
     
         18 . A data analysis method for analyzing target oligonucleotide sequencing data comprising:
 a) scanning a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one target-oligonucleotide hybrid complexes is preferentially cleaved by a nuclease;   b) processing the intensity of the signal wherein a plurality of subsequences are identified;   c) executing a sorting program to sort all of the subsequences based on a defined hierarchy wherein the defined hierarchy sorts the subsequences in order; and   d) determining which subfragments have been found in the target oligonucleotide sequence.   
     
     
         19 . A data analysis system for analyzing target oligonucleotide sequencing data comprising:
 a) a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one target-oligonucleotide hybrid complexes is preferentially cleaved by a nuclease;   b) an automated scanning mechanism wherein the automated scanning mechanism scans the substrate identifying a plurality of subsequences;   c) a sorting program to sort all of the subsequences using a defined hierarchy wherein the defined hierarchy sorts the subsequences and determines, in order, which subfragments have been found in the target sequence.   
     
     
         20 . A computer-readable medium programmed with instructions for analyzing data target oligonucleotide sequencing data comprising computer-executable instructions for causing a programmable processor to execute a method comprising:
 a) receiving a plurality of intensities signals wherein the signal identifies a subsequence from a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one target-oligonucleotide hybrid complexes is preferentially cleaved by a nuclease;   b) sorting the subsequences in order using a defined hierarchy wherein the defined hierarchy sorts and determines, in order which subfragments have been found in the target sequence; and   c) retaining the sorted subsequences in the computer-readable medium.   
     
     
         21 . A method for identifying a base, the method comprising:
 a) hybridizing:
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length, and 
 a target nucleic acid, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; 
   b) hybridizing the target-oligonucleotide probe hybrid complexes with a pool of labeled, ligatable oligonucleotide probes of a preselected length,   c) ligating the labeled ligatable oligonucleotide to the target-oligonucleotide hybrid complex with a ligase;   d) contacting the target-oligonucleotide hybrid complexes with a nuclease under conditions such that the nuclease preferentially cleaves target-oligonucleotide hybrid complexes; and   e) identifying which base has specifically interacted with the target nucleic acid as an indication of a subsequence that is complementary.   
     
     
         22 . The method according to  claim 21  wherein the target nucleic acid is ribonucleic acid (RNA). 
     
     
         23 . The method according to  claim 21  wherein the nuclease is an RNA nuclease. 
     
     
         24 . The method according to  claim 21  wherein the target nucleic acid is deoxyribonucleic acid (DNA). 
     
     
         25 . The method according to  claim 21  wherein the nuclease is a DNA nuclease. 
     
     
         26 . The method of  claim 21 , wherein the array of target; oligonucleotide hybrid complexes has been formed on a substrate comprising beads. 
     
     
         27 . A method for analyzing a target nucleic acid, the method comprising:
 a) providing a reaction system comprising:
 a heating mechanism 
 at least one cavity next to the heating mechanism and 
 a fluorescence detector next to the cavity 
   b) providing a reaction chamber comprising
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length 
 a target nucleic acid 
   c) hybridizing the target nucleic acid to at least one of the oligonucleotides to form target-oligonucleotide hybrid complexes of complementary subsequences of known sequence using the heating mechanism;   d) contacting the target-oligonucleotide probe hybrid complexes with a ligase and a pool of labeled, ligatable oligonucleotide probes of a preselected length,   e) contacting the target-oligonucleotide hybrid complexes with a nuclease under conditions such that the nuclease preferentially cleaves target-oligonucleotide hybrid complexes;   f) positioning the reaction chamber to the cavity such that substrate surface faces the fluorescence detector; and   g) determining which of the oligonucleotides contain the labeled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of the target nucleic acid.   
     
     
         28 . A diagnostic kit comprising:
 a) at least one cavity next to the heating mechanism;   b) a fluorescence detector next to the cavity and d) a reaction chamber;   c) at least one substrate comprising an array of oligonucleotide probes of a preselected length,   d) a target oligonucleotide to be sequenced;   e) a pool of labeled, ligatable oligonucleotide probes of a preselected length;   f) a ligase, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; and   g) a nuclease, wherein the nuclease preferentially cleaves target-oligonucleotide hybrid complexes.   
     
     
         29 . A data analysis method for analyzing target oligonucleotide sequencing data comprising:
 a) scanning a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex, wherein at least one target-oligonucleotide with a labeled oligonucleotide that has not been preferentially cleaved by a nuclease provides an intensity of a signal;   b) processing the intensity of the signal wherein a plurality of subsequences are identified;   c) executing a sorting program to sort all of the subsequences based on a defined hierarchy wherein the defined hierarchy sorts the subsequences in order; and   d) determining which subfragments have been found in the target oligonucleotide sequence.   
     
     
         30 . A data analysis system for analyzing target oligonucleotide sequencing data comprising:
 a) a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex, wherein at least one target-oligonucleotide with a labeled oligonucleotide that has not been preferentially cleaved by a nuclease provides an intensity of a signal;   b) an automated scanning mechanism wherein the automated scanning mechanism scans the substrate identifying a plurality of subsequences;   c) a sorting program to sort all of the subsequences using a defined hierarchy wherein the defined hierarchy sorts the subsequences and determines, in order, which subfragments have been found in the target sequence.   
     
     
         31 . A computer-readable medium programmed with instructions for analyzing data target oligonucleotide sequencing data comprising computer-executable instructions for causing a programmable processor to execute a method comprising:
 a) receiving a plurality of intensities signals wherein the signal identifies a subsequence from a substrate comprising a hybridized array of target-oligonucleotide hybrid complexes of complementary subsequences of known sequence wherein at least one labeled, oligonucleotide is ligated to the complex, wherein at least one target-oligonucleotide with a labeled oligonucleotide that has not been preferentially cleaved by a nuclease provides an intensity of a signal;   b) sorting the subsequences in order using a defined hierarchy wherein the defined hierarchy sorts and determines, in order which subfragments have been found in the target sequence; and   c) retaining the sorted subsequences in the computer-readable medium.   
     
     
         32 . A method for directly synthesis of long oligonucleotides comprising:
 a) forming a hybrid complex by combining at least two oligonucleotides which are phosphorylated at their 5′ ends with a chip-bound oligonucleotide, wherein the chip-bound oligonucleotide having subsequences which are complementary to a subsequence of each of the oligonucleotides;   b) contacting the hybrid complex with a ligase to form a ligated oligonucleotide; and   c) releasing the ligated oligonucleotide from the chip-bound oligonucleotide to form a single-stranded nucleic acid sequence.   
     
     
         33 . A method for directly synthesis of long oligonucleotides, the method comprising:
 a) providing a reaction system comprising:
 a heating mechanism 
 a reaction chamber 
   b) providing a reaction chamber comprising
 a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of at least two oligonucleotides which are phosphorylated at their 5′ ends; 
   c) hybridizing the oligonucleotides which are phosphorylated at their 5′ ends to the oligonucleotides on the substrate to form a hybrid complex;   d) contacting the hybrid complex with a ligase to form a ligated oligonucleotide; and   e) releasing the ligated oligonucleotide from the chip-bound oligonucleotide to form a single-stranded nucleic acid sequence.   
     
     
         34 . A synthesis kit comprising a method for analyzing a target nucleic acid, the method comprising:
 a) a heating mechanism;   b) a reaction chamber; and   c) at least two oligonucleotide to be sequenced wherein their 5′ ends are phosphorylated;   d) a substrate comprising an array of oligonucleotides each of which is complementary to a subsequence of the oligonucleotides with their 5′ ends phosphorylated; and   e) a ligase, thereby forming ligated oligonucleotide.   
     
     
         35 . A method for forming mutant sequences comprising:
 hybridizing:   a) a first oligonucleotide which is phosphorylated at their 5′ end   b) a second oligonucleotide which is phosphorylated at the 5′ end wherein at least the second oligonucleotide has a mutation   c) a substrate comprising an array of oligonucleotides each of which is complementary to a subsequence of each oligonucleotide to form a hybrid complex;   d) contacting the hybrid complex with a ligase to form a ligated oligonucleotide; and   e) releasing the ligated oligonucleotide from the chip-bound oligonucleotide to form a single-stranded nucleic acid sequence.   f) The method according to  claim 1  wherein the mutation is at an internal position of the second oligonucleotide.   
     
     
         36 . The method according to  claim 35  wherein the mutation is near a junction of the second oligonucleotide.