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Rapid Screening of Megadalton mRNA and dsDNA using IMgenius

· mRNA,DNA,Plasmids,Ion Mobility,Stability

Introduction:

The recent global pandemic has highlighted the impact of nucleic acid-based modalities. Messenger RNA (mRNA) and double stranded DNA plasmids are being used in emerging gene and cell therapies. Large sized nucleic acid moieties remain challenging to analyze using traditional LC-MS techniques due to structural heterogeneity, ionization and detection limitations. IonDX™ has developed a bench-top atmospheric pressure ion mobility spectrometer, IMgenius™ to address such challenges. This patented technology was used to profile native mRNA and DNA constructs ranging in sizes from 320kD to 2.83MDa.

IMgeniusTM System:

IMgenius system is a bench-top atmospheric pressure-based ion mobility spectrometer that includes a charge-reducing electrospray ion source. The charge reducing process starts with electrospraying analyte ions dissolved in volatile buffer that generate primary droplets carrying a single analyte ion. These droplets are then charge reduced by an incoming flow of bipolar air ions (shown above on the left). Through gentle droplet evaporation, singly charged ions that are not coulombically stretched out are generated. Such singly charged ions are not distorted and are able to maintain native conformation. Charge reduced analyte ions then get deflected onto detection rings carefully positioned on the wall of the spectrometer. Once ions land, ion mobility spectra are recorded through analog current measurement, thus circumventing the usage of micro channel plates in traditional MS systems. This enables detection of megadalton sized analytes like mRNA without difficulty. 

mRNA Screening:

Ion Mobility spectra of native 3420 bp beta gal mRNA at 50nM shows presence of polydispersity and subpopulation of higher order structures.

Plasmid Screening:

Ion Mobility spectra of native 2.83MDa pBR322 plasmid shows a symmetrical distribution of conformations confirming the presence of a primary supercoiled structure (larger ions in the higher 1/K range). 

mRNA Stability:

Ion Mobility spectra of shear stressed3420bp beta gal mRNA shows a clear degradation with increased number of press cycles with full breakdown happening on the 15th cycle.

Ion Mobility spectra of thermally stressed 996bp EGFP mRNA shows a distinct melting of the construct between 70-75 degrees C which correlates with industry reference.

Ion Mobility spectra of pH stressed 996bp EGFP mRNA shows a clear degradation with higher pH as the mobility spectra shifts completely to the lower 1/K range

Plasmid Stability:

Ion Mobility spectra of shear stressed 2.83MDa pBR322 plasmid shows a stable sample with significant degradation occurring after 20th press cycle

Ion Mobility spectra of thermally stressed 2.83MDa pBR322 plasmid shows a relatively stable sample with melting happening at higher temperature range of 90 degrees C

Ion Mobility spectra of pH stressed 2.83MDa pBR322 plasmid shows a stable sample with very low reaction to changing pH

Analytical Methods:

IMgenius system was used to produce singly-charged ions of 996bp EGFP mRNA (Trilink Biotechnologies), 3420bp beta gal mRNA (Trilink Biotechnologies) and 4361bp pBR322 plasmid (Thermo Fischer Scientific). Samples underwent Ammonium Acetate buffer exchange onto P-6 desalting gel columns (Bio-Rad) prior to infusion. Sample concentration range used was 2.5nM-75nM. Various stability studies were done using heat, shear pressure, pH and other variables like freeze/thaw, dilution (not shown). Ion mobility spectrum of intact nucleic acids were processed using the ION Browser™ software. 

Additional Applications of IMgenius

IonDX’s industry-wide collaborations have allowed us to develop analytical solutions across many additional classes of biotherapeutics, including the characterization of:

  • Non-covalent protein complexes
  • High molecular wieght aggregates
  • mRNA
  • Glycoproteins
  • CRISPR-Cas
  • Virus particles
  • Lipid nanoparticles

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