(T1030-06-36) Sanger Sequencing of Oligonucleotide Pharmaceuticals

Purpose: The Oligonucleotide pharmaceutical industry is a rapidly growing field with a wide variety of drug substances, comprised of nucleic acid chains (DNA, RNA or other modified nucleotide analogues). Recent advancements in CRISPR-CAS technologies have driven interest in longer oligonucleotides (≥100 bases in length). The order of the nucleobases in the chain is essential to the drug’s mechanism. Identity tests confirming the order of the bases are critical to ensure the drug will be effective. Sanger Sequencing is the gold standard in non-mass spectrometry-based approach for determining the order of bases in oligonucleotides. Sanger Sequencing utilizes polymerase chain reaction (PCR) to produce fluorescently labeled sequencing products that are analyzed using capillary gel electrophoresis (CGE). This technique is widely used in the gene sequencing fields but has not been adopted for sequencing synthetic oligonucleotides. One reason for this is because of technical challenges with designing primers (short DNA sequences serving as polymerase start sites). When selecting primers, it is recommended to choose annealing sites ~20 bases away from the region being sequenced, this cannot be done for synthetic oligonucleotides (hereafter referred as “oligo”). To overcome this limitation, we have developed a method where an external primer start site is ligated to the oligo increasing the continuous read length (CRL).

Methods: An oligo comprised of 100 unmodified RNA bases was designed with a variety of base motifs to test the suitability of Sanger Sequencing methods. The oligo had to be reverse transcribed (RT) and PCR amplified before the Sanger Sequencing reaction. For the initial RT-PCR process, two unique internal primers were designed to anneal directly to the oligo. The length of each primer was selected based on the calculated annealing melting temperature (tm) with the oligo, ideal primers have a tm of 50-60⁰C. Primers are incorporated into double stranded copy DNA (cDNA) produced by the PCR process. The inclusion of the primers in the cDNA reduces the possible total sequence coverage achievable by the method. To overcome this limitation, a short oligonucleotide was designed to ligate to the 3’ end of oligo providing a universal primer start site. An external primer start site allows the RT-PCR to be performed with only one overlapping (internal) primer. The use of an external primer start site increases the CRL for oligos and increases the sequence coverage. The sequencing products created during this process were analyzed using CGE and the data for both methods was compared.

Results: Both methods were used successfully to obtain sequence different levels of sequence coverage of the designed oligo. The data interpretation was automated using software provided on the CGE instrument. Using two unique internal primers where 15 bases anneal in the forward direction (tm = 58⁰C) and 17 bases anneal in the reverse direction (tm = 57.3⁰C), 68% of the oligo were verified. 32 out of the 100 possible oligo bases overlap with the primer annealing regions and could not be included in the sequencing results, because primers are included in the cDNA. The 3’ ligation reaction was successfully verified using gel electrophoresis. Incorporating an external start site enabled the RT-PCR process to be performed using one internal primer, the 17 base reverse primer, and an 18 base universal M13 forward primer. Because only 17 primer bases overlap with the oligo, 83% sequence coverage was able to be obtained. The 3’ ligation method has been applied to two different Nitto Avecia client oligonucleotides where 84% and 85% sequence coverage was obtained (raw data not shown for client confidentiality). In all cases different length primers were designed based on the tm being within the desired range.

Conclusion: Sanger Sequencing is an effective method for analyzing oligonucleotide pharmaceuticals. It can used to determine the order of bases in oligonucleotides and successfully implemented for identity tests. We have implemented Sanger Sequencing to confirm the order of ≥80% of the bases in an oligo ≥ 100 bases in length. However, this technique is cable of confirming the order of bases up to 1,000 bases. The maximum theoretical percent sequence coverage is related to the total number of bases in the oligo and the length of the overlapping primers. Sanger Sequencing methods will produce the best results on longer oligonucleotides ≥100 bases. Ligating external start sites allow for longer CRL inside of oligonucleotide and results in higher usable sequence coverage. Future work will be dedicated to ligating a second start site to the 5’ region of the oligo enabling the method to achieve 100% sequence coverage. While this approach has tremendous potential, it should be noted that the Sanger Sequencing approach has the capability to confirm the order of the four canonical bases (A, C, G, T/U), but is insensitive to modifications on the bases, sugars or linkers. Mass spectrometry could be used as a complimentary approach for identifying modifications.