ABSTRACT

1. Modifications to MerMade 192 High Throughput synthesizers for Efficient Aptamer Synthesis Dipak Goyal, Paul J. HatalaArchemix Corp., 300 Third Street, Cambridge MA 02129 Jeff Strauss, Jeff ScheumackBioAutomation Corp., 8408 Kenning Court, Plano TX 75024 RESULTS ABSTRACT Aptamers are oligonucleotides that bind protein targets with high affinity and specificity, thereby disrupting protein-protein interactions. Typically, candidates for pharmaceutical development range in length from 15-40 nucleotides, and can be heavily substituted. However, since the discovery process is SELEX based, the initial synthetic constructs may contain molecules greater than 80 nucleotides in length. In order to facilitate the drug discovery process, high throughput oligonucleotide synthesis is essential. We report the modifications to a MerMade 192 high throughput synthesizer that allow for the parallel synthesis of a wide range of substituted oligonucleotides (15mer to > 80mer). This work is a collaborative effort undertaken by Archemix Corp. and Bioautomation Inc. INTRODUCTION The MM192, Figure 1, is configured with 10 amidite ports with customization up to 20 amidite ports. Each amidite channel is supported with activator channel, ensuring supply of controlled amidite/activator mixture. The ancillary reagent supply can be configured with 12 channels of acetonitrile, deblock, oxidizer, and capping. With these options, the MM192 is not only a high throughput synthesizer, but has the reagent options to make high-quality DNA/RNA/LNA & aptamers. BioAutomation has modified the MM192 software/hardware to fulfill the high degree of complexity in both substitution and size required by Archemix Corporation. Furthermore, in order to obtain highly efficient oligomer synthesis, consistent contact times are required for all lengths of oligonucleotide. BioAutomation has prepared a custom software solution to ensure consistent contact times across a wide variety of chemistries and lengths. METHODS In order to test the software modifications on the MerMade 192, 96 oligonucleotides were synthesized. Starting with a 15-mer, each well added an additional 2’-deoxyribose monomer, ending a 110-mer as shown in Figure 3. The oligonucleotides were cleaved and deprotected under standard conditions and then subjected to high-throughput mass spectrometry and analytical anion exchange HPLC. The results are shown in Table 1 and Figure 5. Figure 3. Schematic of plate showing positions of corresponding lengths of aptamers Table 1. High throughput mass spectrometry results 26Mer 74Mer Maintaining the critical contact time was achieved by a software modification that adds an additional acetonitrile wash for completed columns making each column experience the same vacuum during draining step. Figure 4 illustrates the stepwise addition of reagents. 86Mer 38Mer 50Mer 98Mer 110Mer 62Mer Figure 5. Selected analytical SAX-HPLC Data B A C CONCLUSION Based on the high throughput MS and analytical SAX-HPLC results, the software upgrade provided consistent contact time across a large increase in oligonucleotide length, without loss of purity. ACKNOWLEDGEMENTS The authors appreciate the many important contributions of Jim McArdle. Figure 2. 96 Well plate for high throughput synthesis E D Figure 4. Screen shots depicting stepwise addition of reagents A) Extra acetonitrile wash, B)deblocking, C) Complete acetonitrile wash, D) Extra acetonitrile wash, E) Coupling Figure 1. MerMade 192 synthesizer