The ability to design and synthesize DNA and RNA sequences has had a huge impact on biotechnology particularly in the rapidly growing fields of synthetic biology and nucleic acid-based drug development. Indeed, the use of synthetic DNA/RNA sequences and their analogues for recognition and binding to messenger RNAs encoding disease-causing proteins has led to the production of nucleic acid-based drugs capable of inhibiting the expression of these proteins through either an antisense or an RNA interference pathway in the potential treatment a wide spectrum of human diseases. Such applications require the production of synthetic nucleic acid sequences in large quantities (e.g., millimoles) and high purity for preclinical and clinical investigations. In contrast, total gene synthesis for synthetic biology applications requires small amounts (e.g., nanomoles) of numerous highly pure synthetic DNA sequences. Although the chemical synthesis of nucleic acid sequences using the phosphoramidite chemistry is efficient and can be scaled up for pharmaceutical production, the purification of these sequences presents a formidable challenge.
Despite the fact that the coupling efficiency of phosphoramidite monomers is near quantitative on controlled-pore glass (CPG) support, the full-length nucleic acid sequences are mixed with shorter sequences resulting from incomplete phosphoramidite coupling at each cycle of the nucleic acid sequence assembly. Other process-related impurities consist of deletion sequences due to failure to quantitatively prevent the growth of shorter than full-length sequences and to completely remove the 5′-hydroxyl protecting group at each step of the nucleic acid sequence assembly. Furthermore, the formation of longer than full-length nucleic acid sequences occurs when the activation of phosphoramidite monomers by a weak acid prompts the premature cleavage of the acid-labile 5′-hydroxyl protecting group of the newly extended nucleic acid sequence. Although these impurities are produced in small amounts, their physicochemical similarity to the desired nucleic acid sequence makes them very difficult to remove.
In the context of large-scale nucleic acid-based drug production, HPLC-based methods including reversed-phase (RP) HPLC and anion exchange HPLC are currently the preferred techniques for the purification of nucleic acid sequences. The methods require high-capacity instruments and accessories (e.g., preparative columns) in addition to large volumes of buffered aqueous and organic elution solvents. This process is neither cost-effective nor amenable to parallel purification processes; only a single nucleic acid sequence can be purified per run unless numerous instruments are available for this purpose. HPLC-based purification processes are time-consuming given that, depending on the nature of individual nucleic acid sequence, more than one purification run may be required to achieve the level of sequence purity required for pharmaceutical applications. One important limitation of any large-scale HPLC purification process is the burdensome removal of large volumes of aqueous solvents produced during purification, which may also depend on the physicochemical properties of each nucleic acid sequence; this operation requires costly equipment as well. In regard to the small-scale purification of DNA and RNA sequences for total gene construction in the realm of synthetic biology applications, HPLC-based methods can also be used for this purpose, but as discussed above, these methods are not amenable to cost-effective parallel purification of nucleic acid sequences. Furthermore, HPLC-based purification methods are time-consuming and often may not completely resolve shorter than full-length sequences from the desired nucleic acid sequences. Although polyacrylamide gel electrophoresis (PAGE) can efficiently separate shorter nucleic acid sequences from full length sequences, recovery of the purified sequences from the gel matrix is cumbersome and laborious with limited potential for parallel purification of nucleic acid sequences. A number of orthogonal methods have, however, been proposed for the purification of nucleic acid sequences. These methods are based on affinity, hydrophobic or ion-pair chromatography and solid-phase removal of shorter than full-length nucleic acid sequences by enzymatic hydrolysis or by hydrophobic retention of the full-length sequences.
All of those techniques are either not amenable to highly parallel or large scale purification of nucleic acid sequences or both. Thus, there remains a need in the art for improved methods for the purification of synthetic DNA and RNA sequences.