High-Loading Supports for Oligonucleotide Synthesis

Abstract

b ), and A simple protocol for the preparation of high-loading supports, suitable for large-scale synthesis of oligonucleotides, has been developed. The method involves the use of inexpensive reagents and is amenable to large-scale preparation of supports. The derivatized supports were successfully employed in an automated DNA synthesizer without any difficulty. The quality of the synthesized oligonucleotides was found to be comparable to that of the corresponding oligomers prepared with commercially available standard supports. Introduction. - Synthetic oligonucleotides and their modified analogs have become vital tools for various biological studies. They are finding widespread use in the development of oligonucleotide-based therapeutic and diagnostic tools (1 - 4). Gen- erally, oligonucleotides, in the size range of 15 - 30 residues, are required in abundance, but in tiny quantities for studies like DNA sequencing, etc. However, in case of developing DNA based diagnostics and therapeutics (for use as antisense oligomers), relatively larger quantities of short oligomers are required. To synthesize these molecules in large quantities, the present day methodologies (5 - 7) have proven to be somewhat expensive. Hence, there is a need to develop economically viable routes or protocols for the synthesis of oligonucleotides to meet this exponentially growing demand for these molecules. In the last several years, tremendous improvements have been reported in the area of oligonucleotide synthesis (8) (9). The introduction of new sets of labile protecting groups, fast coupling reagents, and improved polymer supports have revolutionized the entire field so that even a non-chemist can synthesize these molecules quite easily. Since most of the syntheses are currently carried out on solid supports, which play a significant role in deciding the cost of these molecules, there is a need to develop supports having a relatively higher density of the functional groups, which could ultimately reduce the cost of these molecules without compromising the quality of the final product. During the last few years, however, although several polymer supports have been developed and tested, LCAA-CPG (long-chain alkyl- amine/controlled-pore glass) is still the standard polymer support (10) for routine synthesis of oligonucleotides. Recently, we published an express protocol for the functionalization of polymer supports useful for oligonucleotide synthesis (12), in which we reported the loading of LCAA-CPG in the range of 45 - 50 mmol/g of support.