Biology subjects

O'Flaherty, D. K.

Publications and source records attributed to O'Flaherty, D. K..

5 recordsLinked to original sources

bioRxiv2025

A universal buffer system for native LC-MS analysis of antibody-based therapeutics

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful analytical technique for analyzing biological macromolecules. A long-standing challenge has been applying LC-MS at physiological pH under native conditions using volatile buffers. The predominant "buffer" used, ammonium acetate (AmAc, pKa 4.75 for acetic acid and 9.25 for ammonium), does not offer sufficient buffering capacity in the physiological pH range of 7.0-7.4. To address this, we evaluated a set of fluorinated ethylamines, 2-fluoroethylamine (MFEA, pKa 8.9), 2,2-difluoroethylamine (DFEA, pKa 7.2), and 2,2,2-trifluoroethylamine (TFEA, pKa 5.5), that together provide buffering across the 4.5-9.8 pH range. We show that protein separations on strong cation- and anion-exchange resins in these volatile mobile phases perform comparably to traditional non-volatile buffers, with similar elution profiles and analyte elution ranking, albeit with slightly broader peaks. Using fully volatile gradients of pH or ionic strength, we chromatographically resolved charge variants of protein analytes such as mAbs and bovine serum albumin. For many of the eluting LC peaks, we obtained high-resolution mass spectra capable of resolving glycoforms of antibodies. Hydrophobic interaction chromatography (HIC) in volatile mobile phases preserved native separation order and further resolved drug-to-antibody ratio (DAR) species of the antibody-drug conjugate brentuximab-vedotin. For each chromatography modality we further compare innovator and biosimilar antibodies, demonstrating the reproducibility of results in the proposed volatile compounds. Together, our results establish fluorinated ethylamines, in combination with ammonium acetate, as a universal volatile buffer system for native LC-MS, broadly applicable across major chromatographic modalities while maintaining compatibility with mass spectrometry.

biochemistry↗

bioRxiv2025

Lipid acyl chain length and unsaturation modulate membrane surface charge and interactions with amphiphilic DNA nanoprobes

Biological membranes actively regulate their composition to fine-tune their packing, fluidity, phase and surface charge, key properties that influence biomolecular interactions driving essential cellular pathways. While membrane surface charge is often attributed to specific lipid headgroups, the role of acyl-chain chemistry in modulating the interplay between these biophysical membrane properties remains unexplored. Here, we systematically investigate how variations in acyl chain length and saturation modulate lipid packing, fluidity, and membrane surface charge in zwitterionic lipid membranes. Using amphiphilic DNA nanoprobes as model charged biomolecules, we describe the interplay between packing, fluidity, phase and charge, identifying a packing-dependent guiding principle for membrane interactions that persists in the presence of anionic lipids. We also demonstrate that the identity and hydrophobicity of membrane anchors in nanoprobes significantly influence their binding to membranes. By integrating acyl-chain chemistry with membrane biophysical properties into design criteria for biomolecular attachment, our findings provide a mechanistic framework to engineer membrane interactions with both DNA nanoprobes and DNA-based coacervates. Beyond direct application to biomimetic platforms and synthetic cell engineering, these insights are relevant to lipid-based vaccine nanotechnologies and fundamental understandings of membrane-biomolecule interactions in living cells.

biophysics↗

bioRxiv2019

A model for the emergence of RNA from a prebiotically plausible mixture of ribonucleotides, arabinonucleotides and 2’-deoxynucleotides

The abiotic synthesis of ribonucleotides is thought to have been an essential step towards the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabino-nucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabino-nucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

biochemistry↗

bioRxiv2019

Non-enzymatic primer extension with strand displacement

Non-enzymatic RNA self-replication is integral to the RNA World hypothesis. Despite considerable progress in non-enzymatic template copying, true replication remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible solution to this problem in which short invader oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3'-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been catalyzed by short RNA oligonucleotides.

biochemistry↗

bioRxiv2019

Prebiotically Plausible ‘Patching’ of RNA Backbone Cleavage Through a 3′-5′ Pyrophosphate Linkage

Achieving multiple cycles of RNA replication within a model protocell would be a critical step towards demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an RNA world must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an endpoint for RNA replication. Instead, 3' -phosphate terminated RNA strands are able to participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. We found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. Under these conditions, pyrophosphate-RNA can act as a temporary patch to template the polymerization of canonical ribonucleotides, suggesting a plausible non-enzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

biochemistry↗