OligoQuest™

The growing importance of oligonucleotides in research, diagnostics and gene therapy, including tRNA, siRNA, and other modified oligonucleotides up to 120 bases, increased the need for tools to verify their sequence as well as identify and quantify related impurities. Sequence accuracy of RNA and oligonucleotides is critical to ensuring the correct activity and expression as well as avoiding off-target effects or toxicity to patients. However, current Next Generation Sequencing methods do not permit the comprehensive characterization of highly modified RNA-based therapeutics. 

Quadrupole time of flight mass spectrometry (QTOF-MS) instruments are being used to characterize highly modified oligonucleotide sequences as they can determine monoisotopic masses of intact oligonucleotides and their isotopic pattern reliably with high mass accuracy beyond the size of 100mers. 

The intact mass of impurities and side-products of oligonucleotides is not sufficient to infer their identity, therefore information at the nucleotide sequence level is required. MS/MS analyses of synthetic RNA molecules are needed to fully establish the molecular identity of an LC-MS peak. Because oligonucleotide MS/MS spectra are complex, their manual interpretation is time-consuming and requires in-depth analysis skills, rendering the analysis of more than a few samples a day difficult, expensive and often resulting in bottlenecks. 

The OligoQuest™ workflows in BioPharma Compass^® provide the tools to interpret these spectra and to validate the results comfortably and with time-efficiency:

• User defined sequence syntax 
• LC-UV and MS traces are used for quantitation of side products 
  
• MS is used for their High Resolution Accurate Mass (HRAM) determination  

• MS/MS provides sequence specific fragments that the algorithms in OligoQuest™ match to the full-length product (FLP) sequence and putative side products  
  
• OligoQuest™ searches for residue losses, exchanges and additions and reports them 
  
• Strict validation criteria deliver a trustworthy sequence result  
  
• Unique FragCheck mechanism allows to quickly verify the assignment of each fragment ion to the sequence 
  

By exploiting the high isotopic fidelity and mass accuracy of Bruker’s QTOF and timsTOF platforms, OligoQuest™ enables identity confirmation by intact mass and MS/MS analysis. Customers are using OligoQuest™ for confirmation of modified RNA sequences and base exchanges in isomeric oligonucleotides. 

OligoQuest™ offers algorithms and workflows to annotate tandem MS data from oligonucleotide sequences and to identify and quantify side products using the UV peaks.

The OligoQuest™ workflows in BioPharma Compass^® are wizard driven to simplify the method setup and accommodate customer-defined sequence definitions to easily include the wide range of modified nucleotides used by the Pharma industry. 

The OligoQuest™ autoMSMS workflow allows to separate the FLP chromatographically from its side products, determine their monoisotopic molecular weights by high resolution accurate mass analysis, quantify the side products based on their UV traces and identify them by MS/MS. 

The OligoQuest™ targetedMSMS workflow enables a targeted MS/MS analysis to obtain in-depth MS/MS spectra of side products to further confirm the identity of previous uncertain identifications.

 A flexible sequence editor supports natural nucleic acid building blocks as well as custom backbone, base and sugar modifications. Proven algorithms for peak detection and deconvolution of MS and MS/MS data obtained with high isotopic fidelity and ultrahigh resolution ensure correct mass assignments even in high dynamic range samples. 

The timsTOF or QTOF mass spectrometers, paired with the VIP-HESI source for high sensitivity in negative mode, are ideal to analyze oligonucleotide’s sequences such as siRNA but also in larger molecules such as single guide RNA (sgRNA) used in CRISPR based gene editing. 

Confident mass assignments in complex spectra with True Isotopic Pattern and SNAP enable reliable sequence verification even for base substitution resulting in only 1 Da mass differences. 

The confident annotation of complex overlapping spectra is also essential for MS/MS based sequence confirmation. OligoQuest™ combines Bruker’s Maximum Entropy deconvolution and monoisotopic peak picking using the market leading SNAP algorithm with advanced sequencing algorithms from RiboDynamics (embedded in BioPharma Compass^®) to take full advantage of the QTOF’s and timsTOF’s data quality to annotate terminal and internal fragments

Oligonucleotide side-products may include scrambled variants and, e.g., Cytidine-to-Uridine conversions with +1 Da molecular weight differences to the target product sequence, respectively. These can be difficult to characterize solely based on their intact mass. Information at the nucleotide level is required.

Impurities are initially characterized using the OligoQuest™ autoMSMS workflow, which also relies on the accurate isotopic patterns of the intact molecule and its fragmentation. To increase the analysis depth further, the OligoQuest™ targetedMSMS workflow allows to extend the targeted MS/MS analysis time to products of interest and improve ion statistics either by direct infusion or after LC separation. 

The OligoQuest™ workflows in BioPharma Compass^® provide easy to learn tools for the interpretation of oligonucleotide MS and MS/MS spectra. The high isotopic fidelity of Bruker's High-Resolution Accurate Mass (HRAM) QTOF and timsTOF instruments, together with the established SNAP algorithm for monoisotopic peak picking, provide reliable annotation of precursor ions and related low abundant fragment ion masses. MS and MS/MS spectra are thereby matched with high certainty to sequence candidates such as the FLP and expected side-products.