Nucleic Acid detection with Short Prokaryotic Argonautes (NASPA)

Projektbeschreibung

Schnelldiagnostik der nächsten Generation

Die COVID-19-Pandemie hat eine Nachfrage nach schnellen und flexiblen Diagnosegeräten geschaffen. Die Identifizierung bestimmter genetischer Sequenzen ist notwendig für die Pathogenidentifizierung, die Verfolgung von Mutationen und die Diagnose genetischer Erkrankungen. Konventionelle PCR-Tests sind zwar genau, müssen aber in spezialisierten Laboren von geschultem Personal durchgeführt werden, was ihren Einsatz in Kliniken oder im Feld ausschließt. In diesem Kontext zielt das ERC-finanzierte Projekt NASPA darauf ab, kurze prokaryotische Argonauten und synthetische Leit-RNAs zu verwenden. NASPA kann praktisch jede DNA- oder RNA-Sequenz in einer einzigen Reaktion nachweisen und erzeugt bei der Detektion ein Fluoreszenzleuchten. Mit Blick auf die patientennahe Diagnostik und den Einsatz im Feld ist NASPA bestrebt, sensitive, schnelle und flexible Diagnostik bereitzustellen und so die Lücke zwischen der Genauigkeit im Labor und der Verfügbarkeit in der Praxis zu schließen.

Ziel

The SARS-CoV-2 pandemic highlighted the need for versatile diagnostics tools. Detecting presence of specific nucleic acid sequences is a powerful method for pathogen diagnostics, phenotyping, and determination of single nucleotide polymorphisms or other mutations underlying genetic diseases. PCR-based methods, the gold standard in nucleic acid detection, require specialized equipment and trained personnel, limiting its applicability at point-of-care or in-field locations. Alternative isothermal amplification/CRISPR-Cas-based detection methods suffer from technical limitations and a crowded patent landscape.

Nucleic Acid detection with Short Prokaryotic Argonautes (NASPA) is a versatile, sensitive, and specific one-pot nucleic acid detection method that can be used for point-of-care or in-field diagnostics. NASPA builds on our research in which we characterized short prokaryotic Argonaute (pAgo) systems. In NASPA, short pAgos are programmed with synthetic 15-21 nt guide RNAs to detect complementary DNA sequences. Upon detection of the DNA, breakdown of reporter ε-NAD+ is triggered, resulting in a traceable fluorescent signal. As there are no sequence requirements for the guide RNA, and guide RNAs can readily be synthesized, NASPA facilitates detection of virtually any sequence of choice.

We have proof of concept for NASPA-based diagnostics in a laboratory setting (technology readiness level (TRL) 3), and intellectual property has been protected through patent applications. The proposed ERC PoC project has two main objectives 1) enhance NASPA and enable detection of RNA and DNA sequences of economic and/or medical relevance in point-of-care and in-field settings, reaching TLR 5, and 2) build a network of stakeholders including academic and commercial partners to expedite the development of NASPA-based applications and establish a valorisation strategy. Achieving these objectives will pave the way for further development and valorisation of NASPA-based diagnostics.