Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation

Abstract

Interative, preferably computer based iterative processes for generating synthetic compounds with desired physical, chemical and/or bioactive properties, i.e., active compounds, are provided. During iterations of the processes, a target nucleic acid sequence is provided or selected, and a library of candidate nucleobase sequences is generated in silico according to defined criteria. A “virtual” oligonucleotide chemistry is chosen and a library of virtual oligonucleotide compounds having the selected nucleobase sequences is generated. These virtual compounds are reviewed and compounds predicted to have particular properties are selected. The selected compounds are robotically synthesized and are preferably robotically assayed for a desired physical, chemical or biological activity. Active compounds are thus generated and, at the same time, preferred sequences and regions of the target nucleic acid that are amenable to oligonucleotide or sequence-based modulation are identified.

1 . A system of associated components for preparing a set of oligonucleotides and reverse complements that modulate expression of a selected nucleic acid comprising: 
 a computer system that prepares a virtual library of sequences of oligonucleotides and reverse complements targeted to the selected nucleic acid and generates synthesis instructions in computer manipulable form for the sequences of oligonucleotides and reverse complements in the virtual library, wherein the computer system first prepares the virtual library of sequences of oligonucleotides and reverse complements and then reduces the number of sequences of oligonucleotides and reverse complements in the virtual library of sequences of oligonucleotides and reverse complements by one or more of: 
 i) a process of selection based on target accessibility to the selected nucleic acid,  
 ii) a process of selection based on uniform distribution of oligonucleotides and reverse complements across the selected nucleic acid, or  
 iii) a process of selection based on targeting a functional region of the selected nucleic acid;  
   an automated synthesizer that receives the synthesis instructions from the computer system and synthesizes only that set of real oligonucleotides and reverse complements that corresponds to the virtual set of sequences of oligonucleotides and reverse complements consisting of the reduced number of sequences of oligonucleotides and reverse complements; and    an apparatus that accepts the set of real oligonucleotides and reverse complements and performs at least one procedure for each of the real oligonucleotides and reverse complements wherein the procedure identifies particular members of the set that modulate expression of the selected nucleic acid, wherein the procedure is computer-controlled polymerase chain reaction or computer-controlled enzyme-linked immunosorbent assay.    
     
     
         2 . The system of  claim 1  wherein the computer system searches at least one database for alternative transcripts.  
     
     
         3 . The system of  claim 1  wherein the property is modulating the selected nucleic acid.  
     
     
         4 . The system of  claim 1  further comprising a second apparatus selected from the group consisting of liquid chromatography, optical density reader, mass spectroscopy, gel fluorescence and scintillation imaging, and capillary gel electrophoresis.  
     
     
         5 . The system of  claim 1  wherein the functional region is the transcription start site, 5′ cap, start codon, 5′ untranslated region, 3′ untranslated region, stop codon, 5′ splice site or polyadenylation site.  
     
     
         6 . The system of  claim 1  wherein the selected nucleic acid is genomic DNA, cDNA, polymerase chain reaction product, expressed sequence tag, mRNA or structural RNA.  
     
     
         7 . The system of  claim 1  wherein the steps of preparing a set of real oligonucleotides and reverse complements and performing at least one procedure are performed robotically.  
     
     
         8 . The system of  claim 1  wherein each of the at least one property is a physical, chemical or biological property.  
     
     
         9 . The system of  claim 1  wherein the computer network reduces the number of sequences of oligonucleotides and reverse complements in the virtual library of sequences of oligonucleotides and reverse complements by a process of selection based on a uniform distribution of oligonucleotides and reverse complements across the selected nucleic acid.  
     
     
         10 . The system of  claim 1  wherein the computer network applies selected chemical modifications to the virtual oligonucleotides and reverse complements to generate chemically modified virtual oligonucleotides and reverse complements.  
     
     
         11 . The system of  claim 1  wherein the computer network searches at least one database for nucleic acids homologous to the selected nucleic acid.  
     
     
         12 . A method for identifying a oligonucleotides and reverse complement having activity against a target, comprising: 
 generating a set of candidate sequences of oligonucleotides and reverse complements having a predetermined length, wherein the set comprises the set of all sequences having the predetermined length that are complementary to the target sequence;    calculating for the set members at least one of a thermodynamic property score, a sequence property score, and a homology property score;    retaining within the set members having the at least one score within a desired score range;    synthesizing the retained set members; and    assaying the retained set members for activity against the target, wherein the assay is indicative of an amount of an mRNA or a protein encoded by the target.    
     
     
         13 . The method of  claim 12  wherein at least one of the generating, calculating, synthesizing or assaying steps is implemented using a computer.  
     
     
         14 . The method of  claim 12  wherein at least 19% of the assayed set members are not inactive in the assay.  
     
     
         15 . The method of  claim 14  wherein at least 85% of the assayed set members are not inactive in the assay.  
     
     
         16 . The method of  claim 12  wherein the predetermined length is from about 8 to about 30 nucleobases.  
     
     
         17 . The method of  claim 16  wherein the predetermined length is from about 12 to about 25 nucleobases.  
     
     
         18 . The method of  claim 12  wherein the synthesizing step includes use of an automated synthesizer comprising a reagent array delivery format employing motion along a first axis of a matrix of reaction vessels and motion along a second axis of an array of reagents.  
     
     
         19 . The method of  claim 12  wherein the thermodynamic property is selected from the group consisting of a free energy of a target structure, a free energy of an intramolecular-oligonucleotide binding interaction, a free energy of an intermolecular-oligonucleotide binding interaction, a free energy of duplex formation, a free energy of an oligonucleotide-target binding, and a free energy of an alternate thermodynamic property.  
     
     
         20 . The method of  claim 19  wherein the alternate thermodynamic property is a predicted oligonucleotide-target melting temperature.  
     
     
         21 . The method of  claim 12  wherein the sequence property is selected from the group consisting of a number of strings of four G's in a row, a number of strings of three G's in a row, a length of the longest string of A's, a length of the longest string of C's, a length of the longest string of U's, a length of the longest string of T's, a length of the longest string of purines, a length of the longest string of pyrimidines, a percent of A's, a percent of C's, a percent of G's, a percent of U's, a percent of T's, a percent of purines, a percent of pyrimidines, a number of CG dinucleotides, a number of CA dinucleotides, a number of UA dinucleotides, a number of TA dinucleotides, and an alternate sequence property.  
     
     
         22 . The method of  claim 12  wherein the homology property is selected from the group consisting of an homology to a nucleic acid encoding a protein isoform of the target, an homology to an analogous target nucleic acid from a species different from the species from which the target sequence originated, an homology to a splice variant of the target nucleic acid, and an alternate homology property.  
     
     
         23 . The method of  claim 12  further comprising the step of selecting for synthesis a subset of retained set members targeted to a functional region of the target sequence.  
     
     
         24 . The method of  claim 23  wherein the functional region is selected from the group consisting of a transcription start site, a 5′ cap; a start codon, a coding region, a stop codon, a 3′ untranslated region, a 5′ splice site, a 3′ splice site, an exon, an intron, an mRNA stabilization signal, an mRNA destabilization signal, a poly-adenylation signal, a poly-A addition site, a poly-A tail, a gene sequence 5′ of the target pre-mRNA, and an alternate secondary structure property.  
     
     
         25 . The method of  claim 12  further comprising the step of selecting for synthesis a subset of retained set members that are uniformly distributed across the target sequence.  
     
     
         26 . The method of  claim 12  further comprising the step of making a quality control measurement on the synthesized retained set members.  
     
     
         27 . The method of  claim 26  wherein the measurement comprises quantitating an amount of oligonucleotides and reverse complements, determining a percent of total oligonucleotides and reverse complements that is full-length, or determining a mass of total oligonucleotides and reverse complements that is full length.  
     
     
         28 . The method of  claim 27  wherein the measurement comprises a technique selected from the group consisting of ultraviolet spectroscopy, capillary gel electrophoresis, and mass spectroscopy.  
     
     
         29 . The method of  claim 26  further comprising assigning a quality control grade to the synthesized retained set members.  
     
     
         30 . The method of  claim 29  further comprising re-synthesizing the retained set members if the quality control grade is not a passing grade.  
     
     
         31 . The method of  claim 12  further comprising the step of searching a nucleic acid sequence database and selecting for synthesis a subset of retained set members that are not found within the database.  
     
     
         32 . The method of  claim 12  wherein the provided target sequence is selected based on a criterion selected from the group consisting of a quantity of available target nucleotide sequence, a quality of available target nucleotide sequence, an availability of a culturable cell line expressing the target sequence, an availability of a source of reproducible genetic expression of the target, and an association of the target sequence with a disease.  
     
     
         33 . The method of  claim 12  further comprising synthesizing a second retained set wherein the retained set members and the second retained set members differ with respect to an oligonucleotide chemistry, and assaying the second retained set members for activity against the target, wherein the assay is indicative of an amount of an mRNA or a protein encoded by the target.  
     
     
         34 . The method of  claim 34  wherein the modulation is selected from the group consisting of antisense-mediated modulation, RNAi-mediated modulation, and ribozyme-medicated modulation.  
     
     
         35 . A method comprising: 
 receiving an in silico oligonucleotide and reverse complement from an input means;    receiving at least one prescribed property of the oligonucleotide and reverse complement from the input means;    selecting a modification for generating a modified oligonucleotide and reverse complement;    generating in silico the modified oligonucleotide and reverse complement having the prescribed property and selected nucleobase modification; and    communicating the in silico modified oligonucleotide and reverse complement to an output means.    
     
     
         36 . The method according to  claim 35  wherein the step of selecting the nucleobase modification is performed in silico by the computer or is manually performed.  
     
     
         37 . The method according to  claim 35  wherein the input means is at least one computer, which is connected to a network.  
     
     
         38 . The method according to  claim 37  wherein the network is the internet.  
     
     
         39 . The method according to  claim 35  wherein the at least one prescribed property is a base chemistry, sugar chemistry, linker chemistry, or conjugate.  
     
     
         40 . The method according to  claim 35  wherein the modification is a sugar modification, a base modification, a linker modification, or a conjugate modification.  
     
     
         41 . The method according to  claim 35  wherein the output means is a computer or automated synthesizer.  
     
     
         42 . A method comprising: 
 receiving an in silico oligonucleotide and reverse complement to be synthesized having a prescribed property from an input means;    generating synthesis instructions adapted to a synthesizer; and    communicating the synthesis instructions to the synthesizer thereby providing the oligonucleotide and reverse complement to be synthesized.    
     
     
         43 . The method according to  claim 42  wherein the input means is at least one computer, which is connected to a network.  
     
     
         44 . The method according to  claim 43  wherein the network is the internet.  
     
     
         45 . The method according to  claim 42  wherein the at least one prescribed property is a base chemistry, sugar chemistry, linker chemistry, or conjugate.  
     
     
         46 . The method according to  claim 42  wherein the modification is a sugar modification, a base modification, a linker modification, or a conjugate modification.  
     
     
         47 . The method according to  claim 42  wherein the output means is a computer or automated synthesizer.  
     
     
         48 . A method comprising: 
 providing a group of properties which define a target oligonucleotide and reverse complement and from which group a sub-group is selected;    receiving the selected sub-group of properties according to a user selection;    generating in silico, a oligonucleotide and reverse complement having the selected sub-group of properties;    determining in silico, synthesis instructions for the oligonucleotide and reverse complement; and    communicating the synthesis instructions to a synthesizer.    
     
     
         49 . The method according to  claim 48  wherein the group of properties comprises a physical property and a thermodynamic property.  
     
     
         50 . A method comprising: 
 receiving an in silico oligonucleotide and reverse complement to be synthesized having a prescribed property, a threshold criteria, and a user defined target from an input means;    analyzing in silico the oligonucleotide and reverse complement according to the prescribed property, the threshold criteria, and the target thereby producing an analysis report; and    communicating the analysis report to an output means.    
     
     
         51 . The method according to  claim 50  wherein the threshold criteria is a pass fail criteria based on a measure of proper synthetic outcome.  
     
     
         52 . The method according to  claim 51  wherein the 85% proper synthetic outcome is a pass.  
     
     
         53 . The method according to  claim 52  wherein the target is a nucleic acid.  
     
     
         54 . The method according to  claim 50  wherein the target is an RNA.  
     
     
         55 . A method of selecting a oligonucleotide and reverse complement from a library of oligonucleotide and reverse complement according to user selection criterion comprising: 
 receiving a target through an input means according to a user selection;    receiving the user selection criterion, where the user criterion is a property selected from the group comprising a chemical property, a physical property, and a biological property, wherein each property is in relation to the oligonucleotide and reverse complement of the library of oligonucleotide and reverse complement;    obtaining a threshold activity level and a test assay;    comparing a plurality of oligonucleotide and reverse complement of the library to the target according to the test assay, thereby determining a rank for each compared oligonucleotide and reverse complement of the library;    grouping in an active set the oligonucleotide and reverse complement having rank greater than the threshold activity level; and    communicating the active set to an output means.    
     
     
         56 . The method according to  claim 55  wherein the target is a nucleic acid and the selection criteria is a chemical property.  
     
     
         57 . A method of designing a oligonucleotide and reverse complement comprising: 
 receiving a target nucleic acid sequence;    receiving a defined criteria for at least one nucleobase of the oligonucleotide and reverse complement;    receiving a prescribed set of properties comprising a physical property, a chemical property, or a biological property of the oligonucleotide and reverse complement to be designed;    generating a oligonucleotide and reverse complement design according to the prescribed set of properties and the target nucleic acid sequence;    generating in silico a oligonucleotide and reverse complement according to the oligonucleotide and reverse complement design; and    communicating the in silico oligonucleotide and reverse complement to an output means.