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

2021 ◽  
Vol 4 (12) ◽  
pp. e202101213
Author(s):  
Anirudh Chakravarthy ◽  
Anirudh Nandakumar ◽  
Geen George ◽  
Shyamsundar Ranganathan ◽  
Suchitta Umashankar ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Viral Rna ◽  
Diagnostic Assay ◽  
Rna Amplification ◽  
Reporter Protein ◽  
Conformational Switch ◽  
Highly Sensitive ◽  
Luminescence Assay ◽  
Bright Color ◽  
Multiple Variants

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA–based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

scholarly journals Ultrasensitive RNA biosensors for SARS-CoV-2 detection in a simple color and luminescence assay

2021 ◽  
Author(s):  
Anirudh Chakravarthy ◽  
Anirudh K N ◽  
Geen George ◽  
Shyamsundar Ranganathan ◽  
Nishan Shettigar ◽  
...  
Keyword(s):  
Cell Phone ◽  
Rna Amplification ◽  
Reporter Protein ◽  
Human Patient ◽  
Rna Detection ◽  
Conformational Switch ◽  
Diagnostic Strategies ◽  
Highly Sensitive ◽  
Luminescence Assay ◽  
Biosensor Response

The COVID-19 pandemic underlines the need for versatile diagnostic strategies. Here, we have designed and developed toehold RNA-based sensors for direct and ultrasensitive SARS-CoV-2 RNA detection. In our assay, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter-protein e.g. LacZ or Nano-lantern that is easily detected using color/luminescence. This response can be visualized by the human eye, or a simple cell phone camera as well as quantified using a spectrophotometer/luminometer. By optimizing RNA-amplification and biosensor-design, we have generated a highly-sensitive diagnostic assay; with sensitivity down to attomolar (100 copies of) SARS-CoV-2 RNA. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) efficiently detects the presence of viral RNA in human patient samples, with clear distinction from samples designated negative for the virus. The biosensor response correlates well with Ct values from RT-qPCR tests and thus presents a powerful and easily accessible strategy for detecting Covid infection.

scholarly journals Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

2004 ◽  
Vol 70 (5) ◽  
pp. 2632-2638 ◽  
Author(s):  
Claudia Donnini ◽  
Francesca Farina ◽  
Barbara Neglia ◽  
Maria Concetta Compagno ◽  
Daniela Uccelletti ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Kinase Domain ◽  
Arxula Adeninivorans ◽  
Nucleotide Position ◽  
Heterologous Proteins ◽  
Reporter Protein ◽  
Gene Encoding ◽  
Defective Mutant ◽  
Highly Sensitive

ABSTRACT The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

Highly Sensitive Diagnostic Assay for the Detection of Protein Biomarkers Using Microresonators and Multifunctional Nanoparticles

ACS Nano ◽  
2012 ◽  
Vol 6 (5) ◽  
pp. 4375-4381 ◽  
Author(s):  
Jinmyoung Joo ◽  
Donghoon Kwon ◽  
Changyong Yim ◽  
Sangmin Jeon
Keyword(s):  
Diagnostic Assay ◽  
Multifunctional Nanoparticles ◽  
Protein Biomarkers ◽  
Highly Sensitive

scholarly journals Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

2005 ◽  
Vol 79 (9) ◽  
pp. 5743-5751 ◽  
Author(s):  
Jessica E. Petrillo ◽  
Gail Rocheleau ◽  
Brenna Kelley-Clarke ◽  
Lee Gehrke
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Rna Binding ◽  
Alfalfa Mosaic Virus ◽  
Rna Replication ◽  
Comparative Sequence Analysis ◽  
Viral Rna ◽  
Conformational Switch ◽  
Functional Studies ◽  
Functional Features

ABSTRACT Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3′ untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3′ organization model as an alternate model of AMV replication that offers an improved fit to the available data.

P2-038: A highly sensitive diagnostic assay for early diagnosis of Alzheimer's disease

2009 ◽  
Vol 5 (4S_Part_9) ◽  
pp. P273-P273
Author(s):  
Susanne A. Funke ◽  
Lei Wang ◽  
Eva Birkmann ◽  
Franziska Henke ◽  
Oliver Bannach ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Early Diagnosis ◽  
Diagnostic Assay ◽  
Highly Sensitive

scholarly journals Uncoupled Expression of p33 and p92 Permits Amplification of Tomato Bushy Stunt Virus RNAs

1998 ◽  
Vol 72 (7) ◽  
pp. 5845-5851 ◽  
Author(s):  
Sara K. Oster ◽  
Baodong Wu ◽  
K. Andrew White
Keyword(s):  
Viral Genome ◽  
Stop Codon ◽  
Rna Virus ◽  
Rna Replication ◽  
Tomato Bushy Stunt Virus ◽  
Viral Rna ◽  
Rna Amplification ◽  
Viral Rnas ◽  
Reading Frame ◽  
Translational Enhancer

ABSTRACT Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5′-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal ∼6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3′-located segment, region 3.5, was tested. However, when 5′-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5′ cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

scholarly journals Investigation of pooling strategies using clinical COVID-19 samples for more efficient diagnostic testing

2020 ◽  
Author(s):  
Samantha H Adikari ◽  
Emily Z Alipio Lyon ◽  
Attelia D Hollander ◽  
Alina Deshpande ◽  
Elizabeth Hong-Geller
Keyword(s):  
Diagnostic Testing ◽  
Rna Extraction ◽  
Positive Sample ◽  
Viral Rna ◽  
Clinical Samples ◽  
Extraction Column ◽  
Diagnostic Assay ◽  
Significant Difference ◽  
Pooling Strategies ◽  
Single Positive

When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.

A HIGHLY SENSITIVE DIAGNOSTIC ASSAY FOR ALZHEIMER’S DISEASE

2007 ◽  
Vol 2007 (Fall) ◽  
Author(s):  
Susanne Aileen Funke ◽  
Eva Birkmann ◽  
Franziska Henke ◽  
Detlev Riesner ◽  
Dieter Willbold
Keyword(s):  
Diagnostic Assay ◽  
Highly Sensitive

A Rapid Diagnostic Assay for Eastern Equine Encephalomyelitis Viral RNA

1993 ◽  
Vol 49 (6) ◽  
pp. 772-776 ◽  
Author(s):  
Michael H. Vodkin ◽  
Robert J. Novak ◽  
Robert E. Shope ◽  
Gerald L. McLaughlin ◽  
Jonathan F. Day
Keyword(s):  
Viral Rna ◽  
Diagnostic Assay ◽  
Eastern Equine Encephalomyelitis
Sign in / Sign up

Export Citation Format

Share Document