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bioRxiv · 10.1101/2025.04.24.650521

Enhanced protein precipitation with ammonia enables rapid, universal extraction of oligonucleotides for bioanalysis

Kamali, A.·Shelke, J. D.·Sanford, E. J.·Hayashi, M. M.·Chaisakhon, N.·Kolakowski, R. V.·Puri, S.·Zhang, G.
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Abstract

Extracting oligonucleotides from biological matrices for mass spectrometry (MS) using current methodologies is time-consuming and costly. Accelerating the discovery, characterization, and development of oligonucleotides (ONTs) requires a universal method that is low-cost, efficient, without requiring extensive method development for each new test article. Protein precipitation using organic solvents meets these criteria for small molecules but has historically failed for ONTs due to poor recovery arising from their tendency to co-precipitate with proteins. This study introduces an effective approach utilizing small amines dissolved in organic solvents to significantly boost extraction recovery of ONTs from biological matrices .We first present the methods development and then analytically qualify it for the bioanalysis of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) extracted from various tissues, using ion-pairing reverse phase (IPRP) liquid chromatography coupled to tandem MS and high resolution MS (HRMS) detection. This method, Enhanced Protein Precipitation (EPP), achieves near-complete recovery across multiple ONT classes from biological matrices without need of sample digestion, costly solid phase extraction plates, or custom-designed hybridization probes. The method has proven to be more versatile and sustainable than conventional approaches. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/650521v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@a623d2org.highwire.dtl.DTLVardef@179b053org.highwire.dtl.DTLVardef@27273corg.highwire.dtl.DTLVardef@80fe47_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Kamali, A., Shelke, J. D., Sanford, E. J., Hayashi, M. M., Chaisakhon, N., Kolakowski, R. V., Puri, S., Zhang, G.. 2025-04-27. Enhanced protein precipitation with ammonia enables rapid, universal extraction of oligonucleotides for bioanalysis. https://doi.org/10.1101/2025.04.24.650521

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