Results and Discussion
Findings disclose that eluting conditions had a far greater influence on depurination than sequence composition or deblocking conditions. Somewhat surprising was the minimal level of depurination occurring during detritylation. Even several hours following column elution, little depurination of the oligonucleotide had occurred regardless of elution solvent. After ambient overnight incubations, however, results varied considerably as depurination was observed to have drastically increased in nonbuffered pH elutions (water–acetonitrile) using 3% DCA for detritylation (Figure 1).
In stark contrast, sequences eluted in a pH-buffered solvent showed virtually no depurination even when 15% TCA was used for deblocking (Figure 2). The rate of depurination was witnessed to have accelerated among those sequences with a terminal purine when compared to lone internal purine base sequences.
Prior research has observed similar findings, and we postulate that this is the result of the 5´ phosphate group at the apurinic site serving as an electron-withdrawing group further enhancing chain cleavage. When pyrimidine groups were placed at the 5´ terminal, depurination rates diminished considerably. While guanine is reported to release at a slightly faster rate than adenine, our observations yielded no significance difference between the purine bases among the tested sequences. Moreover, adjacent pyrimidine groups were found to have no prevailing influence on depurination.
Today, the majority of synthetic oligonucleotides are produced in combinatorial-style multiplex formats enabling oligo manufacturers to produce tens of thousands of sequences per day. To meet their purification demands, manufacturers are relying on parallel platforms to work in concert with automated liquid-handling systems.
The Clarity QSP product is offered in a 96-well plate that is designed specifically for robotic systems. High-throughput processes can augment depurination as acid exposure times are often longer during detritylation than with cartridge formats. Apurinic occurrence, however, can be avoided simply by using a lower acid concentration during detritylation.
For instance, Figure 3 presents an ESI-MS analysis that shows reducing acid strength to 1% DCA and eluting in a physiological pH buffer averts any depurination and without compromising complete trityl release.
The gathered data presents clear evidence that the primary source for post-column depurination is the pH of the eluting solvent, whereas deblocking conditions and sequence composition are rather minor contributors toward depurination. Further, results clearly show that simple acetonitrile/water mixtures are insufficient to neutralize the eluted oligonucleotide; some buffering to neutral pH is required to prevent depurination.
Our investigation served to address the causes behind the conflicting opinions about trityl-on techniques and depurination. By reducing acid concentration, limiting exposure times, and utilizing physiologically accommodating elution buffers, we were able to substantially minimize, if not eliminate, depurination from occurring during and after on-column detritylation. In addition, sequence composition, considered by many as a primary cause for purine hydrolysis, was discovered to be of little influence under appropriate conditions.
Trityl-on purification has its fair share of opponents and proponents, and this study was designed to evaluate concerns regarding trityl-on purification and its assumed proliferation of depurination. These results clearly show that with a proper protocol, trityl-on purification does not contribute to the degree of depurination of a synthetic oligonucleotide.