Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical Biosensors
Huertas, César S. (Institut Català de Nanociència i Nanotecnologia)
Bonnal, Sophie (Universitat Pompeu Fabra)
Soler Aznar, Maria (Institut Català de Nanociència i Nanotecnologia)
Escuela, Alfonso M. (Universidad de Las Palmas de Gran Canaria)
Valcárcel, Juan (Universitat Pompeu Fabra)
Lechuga, Laura M (Institut Català de Nanociència i Nanotecnologia)

Data:	2019
Resum:	Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.
Ajuts:	Ministerio de Economía y Competitividad TEC2016-78515-R Ministerio de Economía y Competitividad SEV-2017-0706 European Commission 670146 Ministerio de Economía y Competitividad BFU2014-005153 Ministerio de Economía y Competitividad BFU2017-89308-P
Drets:	Tots els drets reservats.
Llengua:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Matèria:	Alternative selections ; Alternative splicing ; Concentration ranges ; Detection accuracy ; Label free analysis ; Label-free optical biosensors ; Quantitative profiling ; Surface plasmon resonance biosensor
Publicat a:	Analytical chemistry, Vol. 91, Issue 23 (December 2019) , p. 15138-15146, ISSN 1520-6882

DOI: 10.1021/acs.analchem.9b03898

Post-print 12 p, 1.0 MB

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