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New method for the detection of RNA viruses such as SARS-CoV-2

Several CIBER-BBN groups at the University of Barcelona, the Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), the Institute of Microelectronics of Barcelona (IMB-CNM-CSIC) and the Institute of Nanoscience and Materials of Aragon (INMA) —a joint institute of the CSIC and the University of Zaragoza (UNIZAR)— have developed a new method for detecting RNA viruses based on the technology of using probes that form triplex structures. This innovative methodology opens up new options to detect viruses such as SARS-CoV-2, the influenza A (H1N1) virus or the respiratory syncytial virus (RSV), a pathogen that affects newborns and requires differential diagnostic care.

This interdisciplinary work, published in the International Journal of Molecular Sciences, is led by Carlos J. Ciudad and Verónica Noé, from the Faculty of Pharmacy and Food Sciences and the Institute of Nanoscience and Nanotechnology (IN2UB) of the University of Barcelona ; Ramón Eritja, Anna Aviñó, Lluïsa Vilaplana and M.Pilar Marco, from IQAC-CSIC and CIBER-BBN; Manuel Gutiérrez, Antoni Baldi and César Fernández, from the IMB-CNM-CSIC, and Valeria Grazu and Jesús Martínez, CSIC researchers at the Institute of Nanoscience and Materials and Aragon INMA (CSIC-UNIZAR) and CIBER-BBN.

The research has counted with the expertise of two NANBIOSIS Units from CIBER-BBN and IQAC-CSIC; NANBIOSIS U2 Customized Antibody Service (CAbS), led by Pilar Marco and Nuria Pascual, and U29 Oligonucleotide Synthesis Platform (OSP), led by Ramón Eritja and Anna Avinó.

This research work was carried out in the context of the PoC4CoV project, led by M. Pilar Marco and César Fernández and financed through the Interdisciplinary Thematic Platform of Global Health of the CSIC. Subsequently, the research has continued as part of a project financed by La Marató de TV3 in 2020 to fight against COVID-19 in which experts from the Faculty of Chemistry of the UB also participate.

Polypurine tweezers to capture viral RNA
The new methodology is based on the ability of polypurine tweezers (PPRHs) —designed by the UB cancer therapy group— to capture viral RNA and form a high affinity triplex. When this hybrid structure is connected to a molecular probe and is brought into contact with the affected patient’s sample, a viral agent detection signal is obtained. The method now presented in the scientific publication has been called the Triplex Enhanced Nucleic Acid Detection Assay (TENADA).

“PPRHs are unmodified single-stranded DNA hairpins that are made up of two mirror domains of antiparallel polypurines. These domains, connected to each other by a thymidine loop, are linked by intramolecular reverse Hoogsteen bonds. Molecular tweezers can specifically bind to polypyrimidine sequences in single-stranded (ssDNA), double-stranded (dsDNA) or RNA viruses through Watson-Crick bonds, thus forming an antiparallel triplex”, details Professor Carlos J. Ciudad, from the Department of Biochemistry and Physiology of the UB.

An effective and faster methodology than the PCR test
Among the advantages that it presents in the detection of viral RNA, it should be noted that the PPRHs methodology can be applied without the intervention of reverse transcriptase —the enzyme that converts RNA into DNA— or the thermocycler (the device that amplifies the material samples). DNA with polymerase chain reaction or PCR). In addition, it has a sensitivity and specificity equivalent to that of the PCR test and can provide results in less than an hour.

In the framework of the work, the team used the hybridization sandwich format in various biosensing devices. This approach uses two oligonucleotides: a triplex-forming PPRH hairpin to serve as the capture probe, and a labeled duplex-forming DNA oligonucleotide to serve as the detection probe.

“The triplex-forming PPRH hairpins were designed to bind to SARS-CoV-2 polypyrimidine sequences, while the detection probes were designed to complement a region near the polypyrimidine target site. Thus, the presence of SARS-CoV-2 RNA is detected by the formation of the ternary complex on the surface of the biosensor”, details Professor Verónica Noé (UB-IN2UB).

This methodology has been implemented in a compact electrochemical device that integrates an electrochemical cell with two electrodes on a chip —manufactured in the Micro and Nanofabrication White Room of the IMB-CNM-CSIC— and a paper fluidic component, and in a Lateral thermal flow implemented in nitrocellulose and using plasmonic nanoparticles and thermal paper that has been developed at the INMA (CSIC-UNIZAR).

TENADA: applications in biomedical research
PPRHs are described in the scientific literature as gene silencing tools for various genes mainly involved in cancer. In addition, they have also been incorporated as probes in biosensors for the detection of small RNA molecules (micro-RNA) to determine the state of DNA methylation and for the diagnosis of pneumonia caused by the fungus Pneumocystis jirovecii.

Now, the new TENADA methodology proves to be effective not only in the detection of viral particles. The high affinity of PPRHs for viral RNA is a property that can be applied to inhibit the virus replication process. For this reason, the antiviral properties of CC1PPRH and CC2PPRH polypurine clamps in cells of the VeroE6 lineage infected with SARS-CoV-2 virions are now also being studied.

In parallel, the work of the different groups involved has also been the basis of a technology that was patented and licensed in July 2022 through the participation of the UB Patent Center, the CSIC and the CIBER-BBN. In turn, this patent has been licensed non-exclusively to the Spanish company Nanoinmunotech through the management of the Bosch i Gimpera Foundation (FBG-UB) in the technology protection process and the company’s license agreement. .

Article of reference:

Aviñó, A.; Cuestas-Ayllón, C.; Gutiérrez-Capitán, M.; Vilaplana, L.; Grazu, V.; Noé, V.; Balada, E.; Baldi, A.; Félix, A.J.; Aubets, E.; Valiuska, S.; Domínguez, A.; Gargallo, R.; Eritja, R.; Marco, M.-P.; Fernández-Sánchez, C.; Martínez de la Fuente, J.; Ciudad, C.J. «Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)». International Journal of Molecular Sciences, diciembre de 2022. Doi:10.3390/ijms232315258

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The coming revolution: synthetic DNA and RNA for therapeutic and diagnostic applications

The discovery of the mRNA vaccines for the treatment of coronavirus, as well as new medicines for the treatment of genetic diseases, has been important in the quest of solutions for undrugable diseases in an unexpected short-time.

Thus, the chemical modifications of nucleic acids with diagnostic and therapeutic purposes is now a reality, a revolution that promises to give hopes on unsolved medical problems or optimize previous approaches, largely due to the research push for the development of mRNA vaccines against SARS-CoV-2 infection.

Now, the prestigious “The Chemical Record” journal has invited the Nucleic Acids Chemistry group, of IQAC-CSIC and CIBER-BBN and belonging to the Global Health Platform of CSIC, to describe the advances and modificacions of the nucleic acids in the last decade. The article, authored by Dr. Carme Fàbrega, Dr. Anna Aviñó and Dr. Ramon Eritja (coordinator and director, respectively, of Unit 29 of ICTS NANBIOSIS), reports the development of synthetic DNA and RNA for therapeutic and diagnostic applications.

The article describes the most important results from the Nucleic Acid Chemistry group in this area covering the international context that surrounded these studies. These include the development of modifications in potentially therapeutic oligonucleotides to enhance nuclease resistance as well as improving cellular uptake and avoiding side effects, and the advances in the use of DNA nanostructures in the controlled deposition of matter in surfaces and their potential application as drug delivery systems is reported.

Moreover, the article has been selected to illustrate the front cover of The Chemical Record, a journal of the Chemical Society in Japan, with a suggestive image that shows the research activity in this area. The back image is the three dimensional reconstruction of a DNA array described by the group of Dr. Seeman obtained by A. Garibotti in Barcelona. On the top the crystal structure of the Argonaute protein from the Protein Data Bank is shown. This protein is a natural player that helps the therapeutic action of RNA molecules. 

Read the full article in this link.

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A project by NANBIOSIS researchers, focused on COVID-19 detection and therapy, selected in ‘La Marató’ research program

The Scientific Commission of ‘La Marató TV3’ has seleced the project entitled “Diagnosis and treatment of Sars-Cov-2 by triplex formation”, led by Ramon Eritja, Scientific Director of NANBIOSIS Unit 29 Oligonucleotide Synthesis Platform (OSP) from CIBER-BBN and IQAC_CSIC to be financed with € 398,750.00. Verónica Noé from the Fundació Bosch i Gimpera Universitat de Barcelona, ​​the CIBERESP researcher Enrique Calderón Sandubete, from the Virgen del Rocío University Hospital and María Valeria Grazú Bonavía, from the Institute of Nanoscience and Materials of Aragón, INMA (CSIC- UNIZE) also participate in this project.

The edition of this solidarity initiative promoted by the Corporació Catalana de Mitjans Audiovisuals, raised more than twelve million euros to finance research against COVID-19. The project was selected, among the 229 submitted, to receive funding for its development.
“Our project wants to solve two important problems in the fight against the COVID-19 pandemic: the first is how to quickly and specifically detect people infected by this virus; and the second is that right now we do not have a effective therapy specifically designed to be used against the virus that causes COVID-19 “explains Dr. Eritja.

The project will try to solve both problems using synthetic oligonucleotides with high and specific affinity for viral RNA. “In recent years, a dozen oligonucleotides have been approved by Health agencies (EMA and FDA) for the treatment of genetic diseases such as Duchene muscular dystrophy as well as metabolic diseases such as familial hypercholesterolemia. In the project we will use specific oligonucleotides, procuced at NANBIOSIS U29, to bind viral RNA that could be used in vitro for the selective capture and diagnosis of the SARS-.COV-2 virus, but also in vivo to inhibit the replication of viral RNA “adds Ramón Eritja.

The call for grants from ‘La Marató’ was carried out, exceptionally, between the months of December and January. Until May, the 229 candidate projects have been reviewed electronically, following a process of proven rigor and transparency, coordinated by the Catalan Agency for Quality and Health Evaluation, in which 95 international evaluators have participated. The assessment, among other aspects, of the quality, the methodology, the scientific, health and social relevance, the innovative value and the viability of the candidate works has determined which projects have been financed with the resources of La Marató 2020.

With this new edition from La Marató, the solidarity project of the Catalan Audiovisual Media Corporation and the Foundation since 1992 an amount of € 216 million have been awarded and a total of 949 research projects hve been promoted, with participation of 9,350 researchers have participated.

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A GLOBAL VISION OF THE COVID-19 PANDEMIC by CSIC

A report from the Spanish National Research Council (CSIC) collects in open access the results of a year of research on SARS-CoV-2. The book ‘A global vision of the COVID-19 pandemic’ shows the responses and solutions obtained by the 300 research teams of the CSIC Global Health Platform. The Institute for Advanced Chemistry of Catalonia (IQAC-CSIC) has participated in the preparation of the chapter ‘Actions in containment and diagnosis’:

Visit CSIC website to read the full news.

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New COVID-19 Diagnostic System Ready for Trials in Clinical Samples

The new system for the rapid, economic and efficient diagnosis of COVID-19 devised by the Universitat Politècnica de València (UPV), the IIS La Fe and the CIBER-BBN is now ready for trials on clinical samples. Since the pandemic broke out, the group of Professor Ramón Martínez Máñez, a researcher at the IDM Institute of the UPV and scientific director of the CIBER-BBN and NANBIOSIS U26 RNM for Biomedical Applications I, has been working on an alternative for the diagnosis of COVID-19, based on nanoporous films. These materials are capable of storing an indicator that is released only in the presence of the SARS-CoV-2 virus thanks to a system of molecular gates. And they do it very quickly, reliably and easily.

The material has already demonstrated its ability to detect the Spike protein present in SARS-CoV-2 and also its function for the detection of a model virus that expresses the aforementioned protein and that has been implemented by the Viral Biology group of the I2SysBio, center mixed of the University of Valencia and the Higher Council for Scientific Research (CSIC), led by Dr. Ron Geller.

“These advances represent another step towards obtaining the final device and confirming that the system is capable of recognizing virus particles”, highlights Ramón Martínez Máñez, director of NANBIOSIS U26 NMR for Biomedical Applications I.

The UPV team of researchers will carry out the clinical validation of the device in collaboration with Dr. Javier Pemán and Dr. Mª Ángeles Tormo, from the Serious Infection Research Group at IIS La Fe, by Dr. María Dolores Gómez, Head of the Molecular Microbiology Unit of the Microbiology Service of the Hospital Universitari i Politècnic La Fe and by Drs. Miguel Salavert and Eva Calabuig, of the Infectious Diseases Unit of the aforementioned hospital.

Once its sensitivity for the diagnosis of COVID-19 has been verified, it is planned to develop a final kit and obtain the CE marking, which will allow its commercialization. “We hope that this process will be carried out as quickly as possible and that it may be on the market to provide a new tool in the face of the pandemic that we are suffering,” says Dr. Elena Aznar, a researcher at CIBER-BBN.

Among the advantages of the device devised by the researchers of the UPV, UV, CSIC, IIS La Fe and CIBER-BBN, I would highlight its ease of use, being able to be used for the diagnosis of COVID-19 in places with limited infrastructure. Furthermore, it does not require highly qualified personnel and the sample does not have to be analyzed in specialized laboratories.

“In addition, the POC technologies on which it is based are global detection tools to monitor for possible new outbreaks in the future, so that once it is ready for use, it will contribute to the rapid implementation of containment measures”, he highlights Ramón Martínez Máñez.

The development of this project is financed by funds from the Covid-19 Donations of the Generalitat Valenciana, made by people and companies during 2020 to contribute to the fight against the pandemic. The solidarity of the Valencian citizenship materialized in donations of about 1 million euros, destined entirely to research projects of excellence against the virus such as the one led by Professor Martínez Máñez, managed by the Ministry of Innovation, Universities, Science and Society Digital.

The project also has the support of the Supera COVID-19 Fund, promoted by Crue Universidades Españolas, Banco Santander -through Santander Universidades- and the CSIC. In addition, the I2SysBio Viral Biology group (UV-CSIC) received funding from the Valencian Innovation Agency (AVI) for the implementation of the model virus.

Related news:

COVID-19 detection system: a fast, cheap and easy to use alternative to PCR.

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NANBIOSIS Unit 2 obtains the Biosafety Level 2 Accreditation

The laboratory of NANBIOSIS Unit 2 from IQAC-CSIC and CIBER-BBN has obtained the biosafety level 2 accreditation, which allows the laboratory to work with biological agents classified in the risk group 2.

Biological containment level 2 (NBS2) laboratories are generally required to work with any derivative of human blood or other primates, body fluids (especially when they are visibly contaminated with blood), cell lines, or tissues in which has uncertainty about the presence of an infectious agent.

Also, the group participates in the COVID project “Point-of-care tests for the rapid detection of SARS-CoV-2 (POC4CoV)”, funded by the CSIC. This project involves the handling of swabs and serum samples from both positive and negative SARS-CoV-2 patients. Lluïsa Vilaplana, member of research group Nb4D of CIBER-BBN and IQAC-CSIC, led by Dra. M. Pilar Marco, wich Coordinates NANBIOSIS U2, has coordinated the process to obtain the accreditation.

The laboratory has an antechamber or clean locker room, a card-controlled entry system and a space for the storage of materials and reagents. It has also an independent air conditioning system, a specific air renewal system and a biosafety cabin type 2A, with HEPA filter. In addition, it is equipped with a suitable lighting system, an emergency lighting system and a large observation peephole on the door.

In addition to this specific equipment, the laboratory is equipped with an inverted microscope, centrifuge, thermostatic bath, stirrers, incubators, refrigerator and autoclave for sterilization and waste management.

The Nanobiotechnology for Diagnosis (Nb4D) research group , focus the research on the development of biomarkers for the diagnosis of infectious diseases. Nowadays, the group participates in five research projects related to this topic. These projects involve working with clinical samples for the detection of the pathogens Pseudomonas aeruginosa (Gram – type bacteria) and Staphylococcus aureus (Gram + type bacteria), both classified in risk group 2.

Source of information: IQAC-CSIC Communication

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Researchers of NANBIOSIS U3 working in a project to develop molecules with neutralizing properties of SARS-CoV-2

The research group of Chemical Multivalent Systems for Nanomedicine and NANBIOSIS U3 Sinthesys of Peptides Unit, of IQAC-CSIC and CIBER-BBN, participates in a new project that seeks to develop molecules with neutralizing properties of SARS-CoV-2, the cause of the COVID-19 disease, for the treatment of patients requiring hospitalization and clinical supervision due to the severity of the infection.

The project, led by the Public University of Navarra (UPNA) and Navarrabiomed, counts with the participation of researchers from institute of advanced chemistry of catalonia (Institute of Advanced Chemistry of Catalonia), IRB Barcelona (Institute for Biomedical Research), CIBER-BBN (Center for Networked Biomedical Research in the thematic area of Bioengineering, Biomaterials and Nanomedicine) and the IRTA (Institute of Agrifood Research and Technology).

With an expected duration of twelve months, the project brings together a multidisciplinary team: chemical synthesis, protein engineering, structural analysis, cell biology, as well as specialists in BSL3 biosafety conditions (the third level of biosafety for laboratories of a scale of four) to be able to test the potential of these molecules.

These molecules with neutralizing properties of SARS-CoV-2 are based on specific peptides of the ACE-2 receptor capable of reducing or nullifying the infectivity of SARS-CoV-2. The chemical part of the project will be carried out by Miriam Royo and Daniel Pulido (NANBIOSIS Unit 3), with the design and performance of the synthesis of ligands based on the ACE-2 receptor.

The consortium partners hope that “the availability of molecules with high inhibitory efficacy will help substantially to mitigate the socioeconomic impact of the pandemic, due to the persistence and / or future outbreaks of SARS-CoV-2 or other potentially dangerous coronaviruses and routes of similar entry ”, in the words of Jacinto López Sagaseta (Navarrabiomed).

The project has been granted in the “Overcome Covid-19 Fund”. This inicitative was created by Crue Spanish Universities, CSIC and Banco Santander with 8.5 million to bust three strategic axes against covid-19: applied research on the virus and its prevention, social impact and strengthening the technological capacity of the universities, and reducing the digital gap. euros. 35 proposals were have been selected out of the 700 submitted.

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New test trials to diagnose Covid 19: Ramon Martinez, Scientific Director of CIBER-BBN is interviewed by Spanish Television

Ramón Martinez, Scientific Director of CIBER-BBN and NANBIOSIS unit 26 NMR: Biomedical Applications II is interviewed by Spanish Television about the research he is coordinating at the Polytechnic University of Valencia to develop new tests as an alternative to PCR.

Dr. Elena Aznar CIBER researcher at IQMA-IDM-UPV group explains how work these test that allow to diagnose quickly, easily, reliably and cheaply if a person is or has been infected by the SARS-COV-2 virus. Ramón Martínez Máñez, leader of the project, reports on the point where the investigation is, as well as the difference of these tests with PCR or antigen tests. These tests implement a technology of the research group that has already been used for other pathogens and that they try to adapt to the COVID virus. “My impression – explains Dr. Martínez – is that the time will come when these tests can be sold in pharmacies and can be used by the users themselves. At the moment we have to see if they work in patient samples and then adapt it so that be a marketable kit by an interested company “

The interview can be whatched here:

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New updated version of the CSIC report on COVID-19

The report by Spanish Higher Council for Scientific Research (CSIC) summarizng the most relevant scientific results on the Covid-19 pandemic.  “A global vision of the Covid-19 pandemic: What we know and what we are investigating from CSIC”, has just published its version number 4.

This report is structured in five blocks: prevention, disease, containment and diagnosis, treatment and vaccines, and global impact.

The containment and diagnosis block describes how the virus is transmitted in the air indoors and the analysis of wastewater as a system for early detection of SARS-CoV-2 is explained. In addition, the importance of diagnosis in the face of the pandemic is underlined and the different types of detection tests available and the diagnostic strategies to control the spread of the infection are detailed. The CIBER-BBN/IQAC-CSIC Nb4D research group and its research platform NANBIOSIS-ICTS Unit 2 Custom Antibody Service (CAbS) takes and important role in this block.

Pilar Marco, Scientific Director of CAbS is co-author, toguether with Cesar Fernandez (IBM-CNM-CSIC) of the chapter 3.4 “The role of the Diagnosis in the face of pandemic” wich provides information on the types of tests, how to perform and how to use them for the different purposes for which  diagnosis is an indispensable tool. “From a broad perspective, we can  distinguish between two types of tests: A. Viral tests: they detect the SARS-CoV-2 virus, include the well-known PCR (Polymerase Chain Reaction) or molecular tests, which detect the genetic material virus B. Test that detect the response of the human body to infection. They include so-called “serological tests, which detect antibodies produced by the patient’s immune system, although other types of biomarkers can also be detected.”

For further information and access to report: http://hdl.handle.net/10261/218312 

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COVID-19 detection system: a fast, cheap and easy to use alternative to PCR.

PCR (acronym in English for “Polymeric Chain Reaction”), is a type of diagnostic test for the detection of infectious diseases. The PCR diagnosis of COVID-19 used since the outbreak of the pandemic offers a high level of specificity and sensitivity but presents a certain degree of complexity, requiring specialized personnel and is expensive.

Researchers from Polytechnic University of Valencia (UPV), the Foundation for the Promotion of Health and Biomedical Research of the Valencian Community (FISABIO), the La Fe Health Research Institute (IIS La Fe) and the consortium Centro de Investigación Biomédica en Red de Bioengineering, Biomateriales y Nanomedicina (CIBER-BBN) has been working, during the pandemic. Within the framework of the Diacovid project, different tests have been carried out with a first prototype of a rapid point-of-care (POC) test, based on nanosystems with molecular gates, that would detect quickly, reliably and easily, SARS-CoV-2.

Ramón Martínez Máñez, Scientific Director of Nanbiosis U26 NMR: Biomedical Applications II explains the advantages of POC techniques as their ability to diagnose in sites with limited infrastructure, without specially qualified staf and without the requirement to transport the sample to a centralized facility. In addition, POC technologies are global detection tools for surveillance against possible new outbreaks in the future. Its use would allow the rapid implementation of containment measures, reduction of therapeutic response times, in situ detection and the use of a low sample volume.

Further information and News in UPV TV

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