SBIR-STTR Award

Solid-phase oligonucleotide synthesis produces a complex mixture that contains, in addition to the target sequence, a plethora of other oligonucleotides. These include a distribution of shorter failure sequences resulting from failed couplings, a heterogeneous mixture of deletion and insertion sequences that are nearly the same length as the target sequence, and branched materials. Purification is necessary to isolate the desired sequence from these other oligonucleotides. For longer sequences, the amount of the by-products becomes larger relative to the target sequence, and purification becomes increasingly difficult. "Trityl-on" purification is a standard technique for short oligonucleotides (20-40 mers). In this approach, nucleobase deprotection and cleavage from the solid support gives a mixture of oligonucleotides, some of which still bear a hydrophobic 5'-dimethyoxytrityl (DMT) group, which allows affinity purification on reverse phase adsorbents, removing non-DMT-bearing failure sequences. Unfortunately, this approach does not easily remove deletion and insertion sequences, which also bear a DMT group. Further, as the oligonucleotide becomes longer, the effectiveness of the trityl-on method diminishes, even for removing failure sequences. Prior attempts to make more hydrophobic DMT analogs have met with some success, but generally involve difficult synthetic work, and none of these building blocks are commercially available. Researchers who require pure oligonucleotides, especially long ones, must still resort to laborious, low-yielding purification methods. The main objective of this proposal is to develop a 5'-protecting group that exploits a different type of affinity interaction, one that is much more powerful than the hydrophobic interaction between a DMT group and reverse phase media. A line of nucleoside phosphoramidites bearing novel 5'-protecting groups is proposed. They will be incorporated into increasingly long oligonucleotides using standard automated protocols, and the resultant "tagged" oligonucleotides will be purified using an alternate type of commercially available adsorbent. After removing the tag, pure oligonucleotides should result, even on long (>100 nt) oligonucleotides. Proposed commercial applications: The proposed affinity-labeled nucleoside phosphoramidites will enable researchers to prepare increasingly lengthy oligonucleotides of unprecedented purity, using technically simple procedures

Solid-phase oligonucleotide synthesis produces a complex mixture that contains, in addition to the target sequence, a plethora of other oligonucleotides. These include a distribution of shorter failure sequences resulting from failed couplings, a heterogeneous mixture of deletion and insertion sequences that are nearly the same length as the target sequence, and branched materials. Purification is necessary to isolate the desired sequence from these other oligonucleotides. For longer sequences, the amount of the by-products becomes larger relative to the target sequence, and purification becomes increasingly difficult. "Trityl-on" purification is a standard technique for short oligonucleotides (20-40 mers). In this approach, nucleobase deprotection and cleavage from the solid support gives a mixture of oligonucleotides, some of which still bear a hydrophobic 5'-dimethyoxytrityl (DMT) group, which allows affinity purification on reverse phase adsorbents, removing non-DMT-bearing failure sequences. Unfortunately, this approach does not easily remove deletion and insertion sequences, which also bear a DMT group. Further, as the oligonucleotide becomes longer, the effectiveness of the trityl-on method diminishes, even for removing failure sequences. Prior attempts to make more hydrophobic DMT analogs have met with some success, but generally involve difficult synthetic work, and none of these building blocks are commercially available. Researchers who require pure oligonucleotides, especially long ones, must still resort to laborious, low-yielding purification methods. The main objective of this proposal is to develop a 5'-protecting group that exploits a different type of affinity interaction, one that is much more powerful than the hydrophobic interaction between a DMT group and reverse phase media. A line of nucleoside phosphoramidites bearing novel 5'-protecting groups is proposed. They will be incorporated into increasingly long oligonucleotides using standard automated protocols, and the resultant "tagged" oligonucleotides will be purified using an alternate type of commercially available adsorbent. After removing the tag, pure oligonucleotides should result, even on long (>100 nt) oligonucleotides. Proposed commercial applications: The proposed affinity-labeled nucleoside phosphoramidites will enable researchers to prepare increasingly lengthy oligonucleotides of unprecedented purity, using technically simple procedures