New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA

Abstract

The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics.

Proposed interactions occurring between arginine–agarose matrix and RNA molecules. Given the multiplicity of arginine side-chain interactions and depending upon RNA folding state, arginine will preferably bind to phosphate groups of RNA backbone or RNA bases.

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Acknowledgments

This work was supported by FCT, the Portuguese Foundation for Science and Technology (PTDC/EBB-BIO/114320/2009 and PEst-C/SAU/UI0709/2011 COMPETE). Rita Martins also acknowledges a fellowship (SFRH/BD/ 64100/2009) from FCT.

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The authors declare no conflict of interest.

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Correspondence to F. Sousa.

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Martins, R., Queiroz, J.A. & Sousa, F. New approach in RNA quantification using arginine-affinity chromatography: potential application in eukaryotic and chemically synthesized RNA. Anal Bioanal Chem 405, 8849–8858 (2013). https://doi.org/10.1007/s00216-013-7334-y

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Keywords

  • Affinity
  • Arginine
  • Chromatography
  • RNA
  • Transcription