key: cord-0770744-ewr1qmj5
authors: Tejedor, Juan R.; Martín, Gabriel; Roberti, Annalisa; Mangas, Cristina; Santamarina-Ojeda, Pablo; Fernández Pérez, Raúl; López, Virginia; González Urdinguio, Rocío; Alba-Linares, Juan J.; Peñarroya, Alfonso; Álvarez-Argüelles, Marta E.; Boga, José A.; Fernández Fernández, Agustín; Rojo-Alba, Susana; Fernández Fraga, Mario
title: Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids
date: 2022-04-25
journal: Anal Chem
DOI: 10.1021/acs.analchem.2c00407
sha: d070a95aad5ff2f1becf0af58810b94317076cf5
doc_id: 770744
cord_uid: ewr1qmj5

[Image: see text] 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.

Excitation wavelengths of 685nm (with 700nm channel) and 785nm (with 800nm channel) were used for Cy5.5

and IRD800 scans, respectively. Densitometric analyses of the corresponding bands were performed with ImageStudio software (LI-COR biosciences).

The FASTA sequences corresponding to 7 human infectious coronaviruses (SARS-CoV-2, SARS-CoV, MERS 

To test the specificity of the FokI-assisted signal amplification assay, different combinations of DNA or RNA oligonucleotides corresponding to orthologous sequences of SARS-CoV-2, SARS-CoV or MERS coronaviruses were tested against different custom dumbbell-like oligonucleotides. Specificity for SARS-CoV-2 was assayed using the Spike dumbbell-like oligonucleotide and related DNA/RNA sequences as for in the basic FokI-assisted signal amplification assay. In addition, a second dumbbell-like oligonucleotide, complementary to MERS Spike protein, but not to other human Beta coronaviruses, was designed and labelled with HEX fluorophore and BHQ-1 

Basic FokI-assisted signal amplification assays: Multicomponent data were downloaded from the instrument and raw data from independent experiments were normalized for potential batch effects using the ComBat function of the R/Bioconductor package sva 5 where Sy represents the standard error of the predicted y-value for each x in the regression and Slope represents the slope value (a) of the calibration plot y= ax + b. The estimation of Sy was performed with the STEYX function according to the following formula:

background normalization was performed by correcting the signal intensities at the different time points against the signal of each corresponding sample at time 0. For each sample, standard scores (z-scores) were calculated according to the normalized fluorescence intensity obtained from 9 validated pre-pandemic negative control cases at a given time point. Samples with z-scores > 2.5 (p < 0.01) were considered to be SARS-CoV-2 positive for viral identification purposes.

Name Code To achieve greater enhancement of the signal amplification properties of the system, we also implemented an additional strategy for signal amplification mediated by a DNA machine, as previously postulated by Weizmann and coworkers 6 

DNA 1 Watson O1 IRD800 - CAGTCGGATGACATGGGTACCATCGTCATC DNA 1 Crick O2 Cy5.5 - CTAGAGATGACGATGGTACCCATGTCATCCGACTGCCTAC RNA 1 Crick O3 Cy5.5 - CUAGAGAUGACGAUGGUACCCAUGUCAUCCGACUGCCUAC DNA 2 Watson O4 IRD800 - GTCAGCCTACTGTACCCATGGTAGCAGTAG DNA 2 Crick O5 Cy5.5 - GATCTCTACTGCTACCATGGGTACAGTAGGCTGACGGATG RNA 2 Crick O6 Cy5.5 - GAUCUCUACUGCUACCAUGGGUACAGUAGGCUGACGGAUG DNA3 Watson O7 IRD800 - TGGGTACCATCGTCATGTCATCCGACGTCGGATGAC MB1 SARSCOV2 (Spike) O8 FAM BHQ1 TGCTGATTCTCTTCCTGTTTCAGCAGTCATCCGACGTCGGATGAC DNA1 SARSCOV2 (Spike) O9 - - CTTGGAACAGGAAGAGAATCAGCAACTGTG RNA1 SARSCOV2 (Spike) O10 - - CUUGGAACAGGAAGAGAAUCAGCAACUGUG Fuel MB1 O11 FAM BHQ1 TGCTGAGCAGCCTCCATCCACTCTGCGCAGAGTGGATGGAGGCTGC MB2 SARSCOV2 (Nsp4) O12 FAM BHQ1 TGCTGATATGTCCAAAGTCAGCAGTCATCCGACGTCGGATGAC DNA2 SARSCOV2 (Nsp4) O13 - - ATTGGTGCTTTGGACATATCAGCATCTATA RNA2 SARSCOV2 (Nsp4) O14 - - AUUGGUGCUUUGGACAUAUCAGCAUCUAUA MB3 SARSCOV2 (ORF8) O15 FAM BHQ1 TGCTGATTTTCTAGCTCCTTCAGCAGTCATCCGACGTCGGATGAC DNA3 SARSCOV2 (ORF8) O16 - - AGAGTAGGAGCTAGAAAATCAGCACCTTTA RNA3 SARSCOV2 (ORF8) O17 - - AGAGUAGGAGCUAGAAAAUCAGCACCUUUA MB1 MERS (Orf5) O18 HEX BHQ1 AGTGAGAACGCATGTCAAACCTCACTGTCATCCGACGTCGGATGAC Control1 DNA SARSCOV O19 - - CATGGGAGAGAAAAAAAATTTCTAATTGTG Control1 DNA MERS O20 - - ATTTCAAGCGTTTGGTTTTTACCAATTGCA Control DNA O21 - - TGCTAGAAAACCGCGTTTCTACGACTGGTG Control1 DNA SARSCOV2 O22 - - ACTTCAGACTATTACCAGCTGTACTCAACT Control2 DNA SARSCOV O23 - - ACCGAAGTTTACTACCAGCTTGAGTCTACA DNA4 MERS (Orf5) O24 - - CCACTGTTTGACATGCGTTCTCACTTTATT Control1 RNA SARSCOV O25 - - CAUGGGAGAGAAAAAAAAUUUCUAAUUGUG

Clustal W and Clustal X Version 2.0

Jalview Version 2--a Multiple Sequence Alignment Editor and Analysis Workbench

Aggressive Assembly of Pyrosequencing Reads with Mates

Software for RNA Secondary Structure Prediction and Analysis

The Sva Package for Removing Batch Effects and Other Unwanted Variation in High-Throughput Experiments

An Autonomous Fueled Machine That Replicates Catalytic Nucleic Acid Templates for the Amplified Optical Analysis of DNA