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Development of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays.

ABSTRACT: Guanine-rich DNA sequences can fold into quadruple-stranded structures known as G-quadruplexes. These structures have been proposed to play important biological roles and have been identified as potential drug targets. As a result, there is increasing interest in developing small molecules that can bind to G-quadruplexes. So far, these efforts have been mostly limited to conventional batch synthesis. Furthermore, no quick on-line method to assess new G-quadruplex binders has been developed. Herein, we report on two new microfluidic platforms to: (a) readily prepare G-quadruplex binders (based on metal complexes) in flow, quantitatively and without the need for purification before testing; (b) a microfluidic platform (based on FRET melting assays of DNA) that enables the real-time and on-line assessment of G-quadruplex binders in continuous flow.

SUBMITTER: Rakers V

PROVIDER: S-EPMC5933291 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Development of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays.

Rakers Viktoria V Cadinu Paolo P Cadinu Paolo P Edel Joshua B JB Vilar Ramon R

Chemical science 20180301 14

Guanine-rich DNA sequences can fold into quadruple-stranded structures known as G-quadruplexes. These structures have been proposed to play important biological roles and have been identified as potential drug targets. As a result, there is increasing interest in developing small molecules that can bind to G-quadruplexes. So far, these efforts have been mostly limited to conventional batch synthesis. Furthermore, no quick on-line method to assess new G-quadruplex binders has been developed. Here ...[more]

PMID: 29780475

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Project description:ObjectivesThe World Health Organization (WHO) had designated the SARS-CoV-2 lineage B.1.1.529 as the new Variant of Concern Omicron (VOC-Omicron) on 26th November 20211. Real-time reverse transcription polymerase chain reaction (RT-PCR), single nucleotide polymorphisms (SNP) and whole genome sequencing (WGS) tests were widely employed to detect SARS-CoV-2 and its variant. Yet, the SARS-CoV-2 Omicron detection performance of commercial real-time RT-PCR platforms and SARS-CoV-2 spike SNP assays remain to be elucidated.MethodsIn the first part of this study, we evaluated the VOC-Omicron detection performance of three commercial RT-PCR sample-to-answer platforms i.e. Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems. The detection performances were compared to one commercial conventional real-time RT-PCR assay (TIB MOLBIOL LightMix Modular SARS and Wuhan CoV E-gene) and one in-house real-time RT-PCR assay targeting RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 in the WHO COVID-19 Reference Laboratory at Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. In the second part of this study, we evaluated the SNP detection performance of four TIB MOLBIOL melting curve-based assays (1. Spike S371L/S373P, 2. Spike E484A, 3. Spike E484K and 4. Spike N501Y) in clinical samples obtained from hospitalized COVID-19 patients in Hong Kong. The SNP results were compared to whole genome sequences generated by Illumina platform.ResultsThe VOC-Omicron detection limits of three commercial sample-to-answer assays were tested to be ≤ 2.35 Log10 dC/ml. The detection performances of the sample-to-answer platforms were comparable to the two tested conventional real-time RT-PCR assays. The test sensitivities of TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P assay and the Spike E484A assays were 100% and 96.6% respectively and the test specificities of both assays were 100%. An aberrant melting peak at Tm 42-44°C was observed when the specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay. Notably, the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike N501Y assay failed to detect the spike N501Y mutation of Omicron variant in the tested specimens.ConclusionsThe SARS-CoV-2 detection sensitivity of three commercial platforms, Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems were shown not to be impacted by the large number of mutations of VOC-Omicron. Also, the signature mutations i.e. Spike S371L/Spike S373P and Spike E484A in VOC-Omicron were correctly identified by the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P and VirSNiP SARS-CoV-2 Spike E484A assays. Unexpected findings including a shifted melting peak or absence of amplification curve/melting peak were observed when specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay and Spike N501Y assay, suggesting a potential alert for Omicron variant, prior confirmation by whole genome sequencing.

Dataset Information

Development of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays.

Publications

Development of microfluidic platforms for the synthesis of metal complexes and evaluation of their DNA affinity using online FRET melting assays.

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