[Slack workspace: #proj-564]

Links to the project:

COVID-19 University of Brasilia Repository

SynBioLab UnB ResearchGate

SynBioLab UnB Website

1.0 Introduction 

1.1 Problem and Background

COVID-19 is a disease caused by the infectious SARS-CoV-2 virus, which started in the province of Wuhan in China in 2019 and quickly spread to all continents. To date, there have been more than 120 million people infected and has already caused more than 2.65 deaths worldwide (https://covid19.who.int/). Brazil is the second country with the largest number of people infected and killed by COVID-19 (https://covid19.who.int/). The detection of infected patients is done mainly by RT-qPCR, or serological methods. Serum diagnostics is based on the detection of IgM and/or IgG and, according to the CDC-USA, its use is not indicated for the diagnosis of the initial phase of the disease (within 7 days from the onset of symptoms). For the initial phase, whose detection is essential to avoid spreading the virus on a large scale and also to define the treatment of the patient, the indicated diagnostics method is RT-qPCR, which identifies the presence of the viral RNA. However, RT-qPCR has several limitations, such as the requirement for skilled labor and equipment, high cost, considerable execution time and laborious procedures. Thus, it is essential to develop new diagnostic methods that may overcome the aforementioned limitations. Today, although the vaccination is advancing around the world, the developing and poor countries present a slow vaccination programme. Moreover, Brazil is going today through its worst moment of the pandemic, and it’s considered the current epicenter of the crisis due to the rise of new variants that might affect the effectiveness of the available vaccines. Thus, the initial detection of the disease will keep being an important action against COVID-19 transmission for a considered period in these countries.

1.2 Solution summary in simple terms

The present proposal aims to develop a fast, sensible, low-cost and easy detection method for the diagnosis of SARS-CoV-2.

The method consists of sampling fluids from the upper airways, RNA extraction, target cleavage reaction, and detection by fluorescence emission. The technical strategies are based on three main steps: the first one consists of the extraction of viral genetic material from a patient and its cut by two designed ribozymes, which are a type of molecular scissors; the second one consists of the connection of DNA probes by affinity to the viral RNA fragment; and the third one is characterised by the augment of red fluorescence emission due to the polymerisation of the probes changing the color of the analyzed solution. In that way, laboratory analysts can distinguish if a patient is infected with SARS-CoV-2 or not.

1.3 Solution summary in technical terms

The first step consists of the recognition of the extracted viral RNA genome by two hammerhead ribozymes, which will be protected initially by thermo-sensitive nanoparticles. The target regions of activity for each of the two ribozymes will be conserved and specific to SARS-CoV-2 sequences obtained after genomic alignment of the different viral strains and its comparisons with genetic material from other SARS and human viruses. This recognition will lead to the cleavage of the viral RNA molecule and the liberation of an initiator fragment. The second step consists of complexing the released viral genomic fragment with two different types of metastable DNA hairpins, containing different fluorophores: one has a donor; and the other, an acceptor. Once the first hairpin matches the viral RNA by complementarity, it will open. Through an overhang, the second hairpin will also open itself and hybridize by complementarity to the first harpin, so on, forming a polymer. Both fluorophores will be close to each other, causing a FRET reaction, which will allow the emission of fluorescence in the wavelength of the acceptor fluorophore. The third stage provides an increase in the fluorescence signal due to the polymerization of the DNA hairpins, so that it increases as a result of the fluorescence of several multiplexed reporter molecules, thus leading to a sensitive method, which would be able to detect small amounts of viral RNA.

1.4 State of advancement of the project

We have already designed and synthesized our ribozymes, and we are currently testing their catalytic activities with a fragment of a synthesized viral RNA, as a proof of concept of our method. We are also improving the designs of the DNA hairpins 1 and 2 in order to optimize and improve the sensitivity and specificity of our FRET-HCR reactions.

1.5 Project Timeline

• [ACHIEVED] Goal 1: Identification of target SARS-CoV-2 sequences for the action of each of the two ribozymes. It is essential that the target sequences have no significant homology to any sequence of other human viruses or to the human genome.
• Goal 1 indicator: identification of at least 3 sites for the performance of ribozymes present in the SARS-CoV-2 genome. This goal was already accomplished.
• [ACHIEVED] Goal 2: Design and synthesis of ribozymes that will act in the cleavage of target sequences identified in goal 1.
• Goal 2 indicator: Obtaining at least 3 ribozymes and the target RNA by in vitro transcription. This goal was already accomplished.
• [PARTIALLY ACHIEVED] Goal 3: Evaluation of the ribozyme cleavage activity through in vitro assay.
• Goal 3 indicator: Obtaining at least 2 ribozymes that cleave the target RNA by in vitro essay. This goal was partially accomplished, since it was observed that, at least, one ribozyme could cleave the target RNA.
• [ONGOING] Goal 4: Design and synthesis of the DNA FRET-HCR hairpins that will be annealed with the initiator fragments obtained after the action of the ribozymes and that will amplify the signal via FRET-HCR reactions.
• Goal 4 indicator: Obtaining at least 4 DNA hairpins that will be annealed to the initiator fragments, and at least 4 DNA hairpins complementary to the first ones that will amplify the system's fluorescence signal. The goal of designing the DNA FRET-HCR hairpins will be accomplished in four weeks, and their synthesis will be performed by a company, and might take two months to be received in Brazil.
• Goal 5: In vitro analysis of our system composed by the viral RNA target sequence, ribozymes, and DNA hairpins.
• Goal 5 indicator: obtaining a fluorescence signal indicating the success of the in vitro operation of the system. This goal will be accomplished in one month after the day we receive the DNA FRET-HCR hairpin molecules. If the test is not successful, the Design-Synthesis-Test cycle will be performed again with other candidate molecules.
• Goal 6: Evaluate if the ribozymes can be protected by thermo-sensitive nanoparticles if needed.
• Goal 6 indicator: obtaining ribozyme cleavage activity after submitting the nanoparticles to high temperatures in order to release their ribozymes cargos. These analyzes will be done during about two months after the first tests have been carried out in goal 4.
• Goal 7:  The essays described above will be performed with samples of RNA molecules obtained from upper airway fluids of infected and not infected (negative control) people, but only once the appropriate conditions have been chosen.
• Goal 7 indicator: obtaining a fluorescence signal intended to be optimized as a simple detection method, indicating the success of the in vitro operation of the system. This final confirmation of the test effectiveness will be done in five months after goal 5 completion.