Enhanced detection of viral RNA species using fokI-assisted digestion of DNA duplexes and DNA/RNA hybrids

Autor: Juan R. Tejedor, Gabriel Martín, Annalisa Roberti, Cristina Mangas, Pablo Santamarina-Ojeda, Raúl Fernández Pérez, Virginia López, Rocío González Urdinguio, Juan J. Alba-Linares, Alfonso Peñarroya, Marta E. Álvarez-Argüelles, José A. Boga, Agustín Fernández Fernández, Susana Rojo-Alba, Mario Fernández Fraga
Přispěvatelé: Instituto de Salud Carlos III, European Commission, Consejo Superior de Investigaciones Científicas (España), Principado de Asturias, Asociación Española Contra el Cáncer, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Centro de Investigación Biomédica en Red Enfermedades Raras (España), Instituto Universitario de Oncología del Principado de Asturias, Tejedor, Juan Ramón [0000-0002-4061-9698], Fraga, Mario F. [0000-0001-8450-2603], Tejedor, Juan Ramón, Fraga, Mario F.
Rok vydání: 2022
Předmět:
Zdroj: Digital.CSIC. Repositorio Institucional del CSIC
instname
Scopus
ISSN: 2018-3682
Popis: The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.
Funding was provided by the ISCIII (COV00624 to J.R.T. andM.F.F., PI18/01527 and PI21/01067 to M.F.F.), CSIC (202020E092 to M.F.F), the European Commission NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) and the Spanish Ministry of Science and Innovation through the Recovery, Transformation and Resilience Plan (GL2021-03-39 and GL2021-03-040), the PCTI from the Asturias Government, co-funded by 2018−2022/FEDER (IDI/2018/146 to M.F.F.), the AECC (PROYE18061FERN to M.F.F), ISPA-Janssen (048-Intramural Nov-Tevar to J.R.T.) and the IUOPA. J.R.T is supported by a JdC fellowship from the Spanish Ministry of Science and Innovation (IJC2018-36825-I). R.F.P. and P.S.O. are supported by the Severo Ochoa program (BP17-114 and BP17-165). A.P. is supported by the PFIS program (ISCIII, FI19/ 00085). J.J.A.L. is supported by the AECC fellowship. C.M. and V.L. are supported by IUOPA, and R.G.U. is supported by CIBERER.
Databáze: OpenAIRE
Externí odkaz: