In Phase I of this grant, synthesis methods suitable for commercial scale preparation of 3'DMT-Nucleosides and their respective amidites and solid supports were developed. Further, the synthetic capabilities of 3'DMT- synthons were demonstrated by synthesis of normal and inversely linked oligonucleotides (ILOs). The availability of 3-DMT-nucleoside synthons and synthesis protocols will allow the chemical synthesis of unique oligonucleotide structures having important applications in the development of novel diagnostic and therapeutic agents and processes. In phase II, a prototype automated synthesis instrument optimized for ILO chemistry will be constructed. The key design feature of the instrument will be a fluidics path which accessed ten or more amidites (4 normal, 4 inverse amidites; 2 linker amidites), and having common low dead volume access to all reagents, resulting in economical consumption of the more expensive 3'DMT synthons. System software will discern the type of bond being constructed (5'-5', 3'-5', or 3'-3') and select the optimal coupling strategy. DMT removal routines will automatically adjust for 3' or 5' DMT deprotection. The instrument will allow multiple synthesis and the generation of combinatorial nucleotide libraries. A commercially operative system will result providing custom synthesis to industrial and academic laboratories of all classes of ILOs. Applications of ILOs as unimolecular primers for PCR, and their use for creation of unique hybridization pairs will be further elements of the Phase II plan.
The current market for DNA synthesis reagents and instrumentation exceeds $100 million and will continue to grow due to expanding markets, especially for PCR technology and antisense therapeutic investigations. The availability of 3'-DMT synthons will add flexibility to the repertoire of oligomer structures which can be synthesized, opening yet another applications frontier for synthetic DNA.
