Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay

Jesper Uhd; Laura Miotke; Hanlee P. Ji; Marina Dunaeva; Ger J. M. Pruijn; Christian Damsgaard Jørgensen; Emil Laust Kristoffersen; Victoria Birkedal; Christina Westmose Yde; Finn Cilius Nielsen; Jonas Hansen; Kira Astakhova

doi:10.1039/D0AN00676A

Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay†

Jesper Uhd,^a Laura Miotke,^b Hanlee P. Ji,^bc Marina Dunaeva,^d Ger J. M. Pruijn,^d Christian Damsgaard Jørgensen,^e Emil Laust Kristoffersen,^f Victoria Birkedal,

^f Christina Westmose Yde,^g Finn Cilius Nielsen,^g Jonas Hansen^ah and Kira Astakhova

*^a

Author affiliations

* Corresponding authors

^a Department of Chemistry, Technical University of Denmark, 207 Kemitorvet, 2800 Kgs. Lyngby, Denmark
E-mail: kiraas@kemi.dtu.dk

^b Division of Oncology, Department of Medicine, Stanford University, 269 Campus Drive, Stanford, California 94305, USA

^c Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, California 94304, USA

^d Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University, Geert Grooteplein Zuid 26 28, 6525 GA Nijmegen, The Netherlands

^e Department of Mathematics and Computer Science, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark

^f Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark

^g Center for Genomic Medicine, 4113 Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

^h Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow, Russia

Abstract

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel “clickable” approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

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Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay†

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Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay

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