Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

Chakravarthy, Anirudh; Anirudh, K N; George, Geen; Ranganathan, Shyamsundar; Shettigar, Nishan; Suchitta, U; Palakodeti, Dasaradhi; Gulyani, Akash; Ramesh, Arati

doi:10.1101/2021.01.08.21249426

Cited by 4 publications

(1 citation statement)

References 45 publications

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“…On the other hand, CRISPR-based methods such as those using the RNA-cleaving Cas13 nuclease ( Joung et al, 2020 ) require relatively expensive, fluorescently labeled RNA-based reporters and additional instruments for fluorescence measurements. Alternative strategies such as PHAsed NASBA-Translation Optical Method (PHANTOM) based on a DNA toe-hold structure coupled to in vitro protein translation and detection ( Chakravarthy et al, 2021 ) require significantly expensive protein synthesis that is in-built into the assay. Therefore, there is a demand for the development of biosensing platforms that possess high specificity and sensitivity, are deployable at a large scale, and are easily adaptable to constantly evolving viral genomes ( Mao et al, 2022 ; Van Ngoc et al, 2022 ; Dong et al, 2023 ; Nelson-Mora et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

BRET-based biosensors for SARS-CoV-2 oligonucleotide detection

Sultana,

Geethakumari,

Islam

et al. 2024

Front. Bioeng. Biotechnol.

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The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype—the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.

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Section: Introductionmentioning

confidence: 99%

BRET-based biosensors for SARS-CoV-2 oligonucleotide detection

Sultana,

Geethakumari,

Islam

et al. 2024

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

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COVID-19 Diagnosis: A Comprehensive Review of Current Testing Platforms; Part B

Fatemi

Nejad

Siadat³

et al. 2021

Covid-19

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Compact RNA sensors for increasingly complex functions of multiple inputs

Choe,

Andreasson,

Melaine

et al. 2024

Preprint

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Designing single molecules that compute general functions of input molecular partners represents a major unsolved challenge in molecular design. Here, we demonstrate that high-throughput, iterative experimental testing of diverse RNA designs crowdsourced from Eterna yields sensors of increasingly complex functions of input oligonucleotide concentrations. After designing single-input RNA sensors with activation ratios beyond our detection limits, we created logic gates, including challenging XOR and XNOR gates, and sensors that respond to the ratio of two inputs. Finally, we describe the OpenTB challenge, which elicited 85-nucleotide sensors that compute a score for diagnosing active tuberculosis, based on the ratio of products of three gene segments. Building on OpenTB design strategies, we created an algorithm Nucleologic that produces similarly compact sensors for the three-gene score based on RNA and DNA. These results open new avenues for diverse applications of compact, single molecule sensors previously limited by design complexity.

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Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

Cited by 4 publications

References 45 publications

BRET-based biosensors for SARS-CoV-2 oligonucleotide detection

BRET-based biosensors for SARS-CoV-2 oligonucleotide detection

COVID-19 Diagnosis: A Comprehensive Review of Current Testing Platforms; Part B

Compact RNA sensors for increasingly complex functions of multiple inputs

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