Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays.

Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5'-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.

The Immunoprobe Aggregation State is Central to Dipstick Immunoassay Performance.

As new infectious disease outbreaks become more likely, it is important to be able to develop and deploy appropriate testing in time. Paper-based immunoassays are rapid, cheap, and easy to produce at scale and relatively user friendly but often suffer from low selectivity and sensitivity. Understanding the molecular mechanisms of paper immunoassays may help improve and hasten development and therefore production and market availability. Here, we study how the behavior of nanoparticle-antibody immunoprobes in paper dipstick immunoassays is impacted by synthesis strategy and surface chemistry architecture. We conjugate gold nanoparticles to polyclonal anti-immunoglobulin G (IgG) and anti-zika NS1 antibodies by electrostatic adsorption and N-hydroxysuccinimide (NHS) and hydrazide (Hz) chemistries. The immunoprobes were used in paper immunoassays and the effective affinity for the antigen was quantified from the test line intensities, as well as the distribution of the immunoprobes throughout the strips. The results show that nanoparticle colloidal stability, both post synthesis and during antigen binding, is a key factor and affects immunoassay results and performance, often through reduction or loss of signal.