Apparatus for combinatorial synthesis

Abstract

Apparatus and methods are described for split synthesis combinatorial chemistry that provides candidate libraries where an even distribution of theoretical products is obtainable through even mixing during the pooling step, followed by controlled redistribution of the mixed pooled products from the prior addition step into separate synthesis columns, one for each different specie of subunit to be added.

1 : A method for generating a library of random sequence oligomers, comprising the acts of:
 evenly distributing a pool of activated or prederivitized fluidized solid supports into each of a plurality of synthesis chambers wherein at least one individual chamber is established for each different monomer subunit species to be included in the random sequence oligomers,   simultaneously conducting a random position addition reaction in each synthesis chamber whereby a random position monomer subunit is added to the activated or prederivitized fluidized solid supports to create a different sequence reaction product in each synthesis chamber;   mixing and pooling the different sequence reaction products by conveying the different sequence reaction products from each synthesis chamber through a static mixer and into a mixing chamber;   redistributing, substantially evenly, the pooled products back into each synthesizer chamber;   conducting a next addition reaction in each synthesis chamber to add a next subunit to the random sequence oligomer; and   repeating the acts of mixing and pooling, redistributing and conducting to produce an oligomer of a defined number of subunits.   
     
     
         2 : The method of  claim 1 , wherein the random sequence oligomer is an aptamer and the prederivitized fluidized solid support comprises a defined PCR primer sequence. 
     
     
         3 : The method of  claim 2 , wherein the aptamer is a thioaptamer. 
     
     
         4 : The method of  claim 1 , wherein the addition reaction is conducted by phosphoramidite chemistry. 
     
     
         5 : The method of  claim 1 , wherein the monomer subunits are phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S 2 -ODNs). 
     
     
         6 : The method of  claim 2 , wherein the defined PCR primer sequence is created in situ in at least one of the synthesizer columns following by conveyance through the static mixer and mixing chamber prior to being evenly distributed into each synthesizer chamber prior to conducting the random position addition reactions. 
     
     
         7 : The method of  claim 2 , wherein the defined PCR primer sequence is created in situ in a separate synthesizer column followed by even distribution into each synthesizer chamber prior to conducting the random position addition reactions. 
     
     
         8 : The method of  claim 1 , further comprising one or more additions of subunits without a mixing and pooling step, thereby generating one or more regions of defined sequence in addition to one or more regions of random sequence. 
     
     
         9 : The method of  claim 1 , wherein each chamber includes fritted reagent inlet and outlet apertures that are adapted to contain fluidized solid supports within the columns during synthesis and wherein each chamber further includes at least on non-fritted mixing aperture through which the fluidized supports are conveyed out of the chambers for pooling and mixing. 
     
     
         11 : The method of  claim 1  wherein the steps of the method are automated and controlled by a programmable controller. 
     
     
         12 : The method of  claim 1 , wherein the static mixer comprises a conduit intersected by fixed elements that create turbulence in the pressurized stream conveying the solid supports through the conduit. 
     
     
         13 : The method of  claim 12 , wherein the fixed elements comprise one or more bars intersecting the conduit. 
     
     
         14 : The method of  claim 12 , wherein the fixed elements comprise one or more helical elements longitudinally fixed in the conduit. 
     
     
         15 : The method of  claim 1 , wherein the mixing chamber is configured by elimination of sharp interior angles to limit trapping of the fluidized solid supports. 
     
     
         16 : The method of  claim 1 , wherein a path of fluid flow through the synthesis chambers is established by action of at least one pressure controllable fluid reservoirs. 
     
     
         17 : The method of  claim 11 , wherein the path of fluid flow through the synthesis chambers is established by action of at least two pressure controllable fluid reservoirs which are disposed at essentially opposite ends of the fluid flow path. 
     
     
         18 : The method of  claim 1  wherein the solid support is selected from the group consisting of inorganic or organic materials including: controlled pore glass (CPG), glass, silica or silica containing materials, carbon, metals, polymers, plastics, and resins. 
     
     
         19 : A method of generating a bead bound library of random sequence thioaptamers, wherein the library is generated by a process comprising the acts of:
 establishing a plurality of individual synthesis chambers, wherein at least one synthesis chamber is established for each subunit monomer species to be included in the random sequence aptamers;   distributing a pool of activated or prederivitized fluidized beads into each synthesis chamber, each bead having a plurality of sites for synthesis of an oligonucleotide;   simultaneously conducting an addition reaction in each synthesis chamber to add an oligonucleotide subunit to the activated or prederivitized fluidized beads;   mixing and pooling the reaction products from each synthesis chamber by conveying the reaction products from each synthesis chamber through a static mixer and into a common mixing chamber;   redistributing, substantially evenly, the pooled reaction products back into each synthesizer chamber;   repeating the acts of mixing and pooling, redistributing and conducting to produce a bead bound library of random sequence thioaptamers, wherein the thioaptamer is generated by programmed addition of phosphorothioate oligonucleotide (S-ODN) or phosphorodithioate oligonucleotide (S 2 -ODN) subunits at defined positions during the synthesis; and   obtaining the bead bound library of thioaptamer from the synthesizer chambers.   
     
     
         20 : The method of  claim 19 , wherein each synthesizer chamber comprises a reagent fluid path selectively controlled by individual inlet and outlet reagent valves disposed on opposite ends of each chamber, and a mixing fluid path selectively controlled by individual first and second mixing valves disposed on opposite ends of each chamber.