Technical Field

The invention relates to a RNA-based riboregulatory biosensor which is suitable for use in diagnostic devices enabling to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen, that causes the global pandemic of coronavirus disease 2019 (COVID-19), and to a method of obtaining thereof.

Background of the Invention

COVID-19 is an infectious disease caused by SARS-CoV-2. It was first reported on the date of December 2019 and spread all over the world rapidly, and caused a pandemic. The virus leading to this disease causes human deaths significantly. One of the most significant factors for reducing and controlling the said deaths is to detect the disease early, reliably and through the use of common diagnostic methods. Thus, use of cost-efficient, quich and portable diagnostic devices is preferred in terms of speeding up the process of controlling the disease. There is a need for a RNA-based riboregulatory biosensor which enables to effectively detect SARS-CoV-2 pathogen in related devices and comprises gene-specific structures in SARS-CoV-2 genome, and a method of obtaining the same.

The United States patent document no. US2019071737, an application in the state of the art, discloses a RNA-based riboregulator used for detection of nucleic acids of viruses. The riboregulator enables to detect the response to a stimulus. The riboregulator comprises the key RNA and the key RNA that triggers it. The key RNA includes a ribosome binding region (RBS), a start codon (AUG), a linker region and a green fluorescent region (GFP). When the specified virus genome is present in the medium, the trigger RNA having the complementary sequence A* binds to the sequence A in the key RNA and the stem of the key RNA is degraded and thus the start codon (AUG) is unlocked with RBS. Following the unlocking, signal increase occurs in the medium by means of GFP.

Summary of the Invention

The objective of the present invention is to realize a RNA-based riboregulatory biosensor which is suitable for use in diagnostic devices enabling to detect SARS- CoV-2 pathogen that causes the COVID-19 disease -a global pandemic- and a method of obtaining thereof.

Detailed Description of the Invention

“A Riboregulatory Biosensor Detecting SARS-Cov-2 Pathogen and A Method of Obtaining Thereof’ realized to fulfil the objective of the present invention is shown in the figures attached, in which:

Figure 1 is a view of the working mechanism of the inventive RNA-based riboregulatory biosensor.

Figure l is a flow chart of the method of obtaining the inventive biosensor.

The components illustrated in the figures are individually numbered, where the numbers refer to the following:

1. RNA-based riboregulatory biosensor

2. Key RNA

21. Key identification region

22. Ribosome binding region

23. Binding region

24. Green fluorescent signaling protein gene region 3. Key RNA

100. Method

R: Ribosome

The inventive RNA-based riboregulatory biosensor (1) which suitable for use in diagnostic devices enabling to detect SARS-CoV-2 pathogen that causes COVID- 19 comprises

- at least one key RNA (2) with the secondary structures which are the key identification region (21), the ribosome binding region (RBS) (22), the linker region (23) and the green fluorescent signaling protein (sfGFP) (24) that has the gene sequence of the protein selected for the SARS-CoV-2 genome; and

- at least one key RNA (3) which has two virus-specific regions in the ORF lab and S genes in the SARS-CoV-2 genome, unlocks the key RNA (2) structure and then makes it ready for ribosome binding.

The key RNA (2) included in the inventive RNA-based riboregulatory biosensor (1) comprises the key identification region (21) enabling to identify the key RNA (3) structure, the ribosome binding region (22) enabling the ribosome (R) -that is responsible for the conversion of mRNA molecules into protein- to bind to the key RNA (2), the binding region (23) enabling to initiate the translation of the target gene, and the green fluorescent signaling protein (sfGFP) gene region (24) releasing a green fluorescent signal that is measurable in the presence of ORF lab and S genes in the SARS-CoV-2 genome. The key RNA (2) prevents access by the ribosome (R) to the ribosome binding region (22) in the absence of the key RNA (3) in the medium and to the binding region (23) that initiates the translation of the target gene, and thus the expression of the target gene is prevented due to the fact that the ribosome (R) cannot to bind to the mRNA molecule. The key RNA (2) ensures that the target gene is identified by means of the key identification region (21) when key RNA (3) is present in the medium the key structure is unlocked and becomes activated, the ribosome (R) binds to the mRNA molecule upon the structure is unlocked, and the mRNA molecule is converted into protein and the measurable fluorescent signal is released.

The key RNA (3) included in the inventive RNA-based riboregulatory biosensor (1) is configured to have two virus-specific genes in the ORF lab and S genes in the SARS-CoV-2 genome.

The inventive method (100) of obtaining RNA-based riboregulatory biosensor (1) comprises the steps of

- selecting the gene regions that can be used as the trigger RNA from the SARS-CoV-2 virus genome (101);

- creating the key RNA (3) structures for the determined regions (102);

- eliminating the non-specific ones for the SARS-CoV-2 from the created key RNA (3) structures (103);

- preparing the key RNAs (2) suitable for the selected key RNA (3) regions and obtaining the biosensors comprising these key RNAs (2) (104).

In the step of selecting the gene regions that can be used as the trigger RNA from the SARS-CoV-2 virus genome (101) of the inventive method (100) of obtaining the RNA-based riboregulatory biosensor (1), the gene regions that can be used as the trigger key RNA (3) are selected in accordance with the criteria for proliferation by isothermal amplification methods by using NCBI: Primer Blast program in the preferred embodiment of the invention.

In the step of creating the key RNA (3) structures (102) of the inventive method (100) of obtaining the RNA-based riboregulatory biosensor (1), the key RNAs (3) are created for the selected candidate trigger key RNA (3) regions by using the NCBI:Primer Blast program in the preferred embodiment of the invention. In the step of eliminating the non-specific ones for the SARS-CoV-2 from the created key RNA (3) structures (103) of the inventive method (100) of obtaining the RNA-based riboregulatory biosensor (1), the key RNAs (3) created are compared with the human genome and the genomes of other human coronaviruses, and the key RNAs (3) having the same or similar sequence with these organisms are eliminated. Also, an elimination is performed based on the secondary structures of the determined key RNAs (3) and the stability of these structures.

In the step of preparing the key RNAs (2) suitable for the selected key RNA (3) regions and obtaining the biosensors comprising these key RNAs (2) (104) of the inventive method (100) of obtaining the RNA-based riboregulatory biosensor (1), de novo key RNA (2) sequences are obtained by using the 'multiple design' option in the NUPACK software for the candidate trigger RNAs having the highest scores from the selected criteria, by excluding the candidates with Gibbs free energy levels below -25 by using NUPACK software of the candidate key RNAs (3) selected after the elimination procedures performed in the preferred embodiment of the invention. The RNA-based riboregulatory biosensors (1), which are used in the detection of SARS-CoV-2 virus by combining the obtained key RNA (2) and the key RNA (3) fragments, are obtained.

The de novo key RNAs (2) obtained by the inventive method (1) specific to the ORFlab and S genes in the SARS-CoV-2 genome are included in Table 1.

Table 1. The de novo key RNAs (2) obtained specific for the ORFlab and S genes in the SARS-CoV-2 genome

The RNA-based riboregulatory biosensor (1) obtained by the inventive method (100) can be used in portable diagnostic devices enabling cost-efficient and rapid diagnosis of SARS-CoV-2 virus thatcauses COVID-19 disease.

Within these basic concepts; it is possible to develop various embodiments of the inventive “Riboregulatory Biosensor (1) Detecting SARS-Cov-2 Pathogen and A Method of Obtaining Thereof (100)”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.