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Projects

PRIN 2022

CRISPR-Cas-based sensing platforms for the monitoring of clinically relevant antibodies

Role: Co-Principal Investigator.

Co-led by Prof. Alessandro Porchetta, University of Rome Tor Vergata.

Funding body: Italian Ministry for University and Research

 

Monitoring and quantifying clinically relevant antibodies as biomarkers for medical conditions and therapeutic regimens is essential for making informed clinical decisions. Current laboratory technologies, such as ELISA and CLIA, along with rapid point-of-care (POC) tests, have limitations that hinder their effectiveness. To address this issue, we aim to develop novel strategies for antibody detection that combine rapid POC analysis with high sensitivity and specificity, thereby paving the way for advanced serological testing platforms.

Our project revolves around leveraging the groundbreaking CRISPR-Cas technology, which has revolutionized genome editing and nucleic acid diagnostics. Our overarching objective is to reengineer and adapt CRISPR-Cas-based detection strategies for the specific detection of biomarker antibodies. These strategies will be seamlessly integrated into electrochemical and optical sensing platforms, enabling the rapid, ultrasensitive, and cost-effective detection of specific antibodies, particularly tumor-associated autoantibodies. These autoantibodies hold great promise as diagnostic and prognostic markers in the field of oncology.

The core of our innovation lies in engineering antibody-responsive nucleic acid-based devices, which will work in conjunction with tailored bioresponsive elements and various Cas enzymes. Our project’s key strategy involves employing rationally designed antibody-controlled nucleic acid systems as molecular translators. These systems convert the binding of a specific antibody into an arbitrary DNA/RNA input, thereby activating a CRISPR-Cas platform. This platform serves as an amplifier, providing an enhanced optical or electrochemical signal.

Our interdisciplinary approach draws from the fields of DNA nanotechnology, biomolecular engineering, and synthetic biology. By uniting these disciplines, we intend to deliver transformative technologies that enhance the efficacy and efficiency of antibody monitoring across a wide range of medical needs.

GUIDO BERLUCCHI FOUNDATION MINI-GRANT PROGRAMME 2022

DNA-Based Molecular Sensors for the Analysis of Oncogenic Zinc Fingers - “DNA-FINGERS”

Role: Principal Investigator.

Funding body: Guido Berlucchi Foundation

https://fondazioneberlucchi.org/it

Identifying molecular patterns and expression profiles associated with cancer is instrumental in translating biological insights into innovative molecular diagnostics and precision medicine therapeutics. Transcription factors (TFs) hold a pivotal role in gene regulation and various vital cellular processes. Their expression often goes awry in cancer cells, making them potential biomarkers for early diagnosis and promising targets in precision therapeutic approaches. Among these, Zinc Finger Transcription Factors (ZNFs) represent the largest TF family, known for their ability to bind specific double-stranded DNA sequences. Numerous studies have linked the abnormal expression of certain ZNFs to cancer onset and progression through diverse mechanisms.

Currently, the detection of ZNFs in biological samples relies on labor-intensive, time-consuming, and expensive laboratory techniques like Western Blot and ELISA. In contrast, in this project, we propose an innovative approach that harnesses the unique DNA-binding activity of ZNFs. Their precise binding to defined dsDNA motifs offers an opportunity to design DNA-based molecular sensors. This can be exploited to design synthetic DNA switches capable of changing conformation upon binding, enabling the engineering of sensing probes and TF-controlled nanomachines for synthetic biology applications. This binding activity-based strategy has recently inspired specific examples of DNA-based translators for TF-controlled DNA circuits yet has to be explored for designing ultra-sensitive technologies to analyze oncogenic ZNFs.

In this project, we propose to develop DNA-based systems that recognize specific oncogenic ZNFs and convert this binding event into an amplified optical or electrochemical signal. To achieve this, we plan to leverage tools and strategies from the field of DNA nanotechnology, where synthetic DNA serves as a programmable engineering material. Through the integration of complementary mechanisms, including new approaches involving cutting-edge CRISPR-Cas systems, we aim to create novel classes of molecular technologies that can be incorporated into optical and electrochemical devices. This innovation will facilitate the analysis of specific oncogenic ZNFs in biological samples, harnessing their inherent DNA-binding activity.

FIL Giovani 2022 – Seed Grant for Young Researchers

Theranostic nucleic acid-based nanodevices artificially regulated by proteolytic enzymes

Role: Principal Investigator.

Funding body: University of Parma and CARIPARMA Foundation.

 

Precision medicine aims to overcome the limitations of traditional diagnostics and therapeutics by targeting specific molecular markers associated with diseases. A promising category of precision medicine tools comprises dynamic molecular devices capable of translating biorecognition events into the activation of programmable molecular processes. These molecular devices offer precise spatiotemporal control and conditional activation, enabling the creation of innovative programmable theranostics with enhanced targeted and localized effects, combining both high specificity and sensitivity. DNA nanotechnology has made significant contributions to this endeavor. Synthetic DNA-based nanodevices, inherently programmable and versatile, can be tailored into customized diagnostics and therapeutics based on predictable base-pair interactions.

A more substantial challenge lies in developing dynamic DNA-based theranostics responsive to target proteins. Achieving this necessitates the establishment of artificial protein-DNA communication and the implementation of non-trivial binding-induced mechanisms. Current methods, relying on proximity effects or conformational changes, only address a fraction of clinically relevant proteins with DNA-based devices, leaving behind other crucial protein families, including proteolytic enzymes. These enzymes could potentially serve as specific endogenous triggers for the controlled activation of DNA-based nanodevices. Of particular interest are metalloproteinases, essential markers in cancer research due to their aberrant expression in various types of cancer. Enabling these proteins to interact with nucleic acid-based systems could open new avenues for precision medicine strategies.

In response to this challenge, this project proposes the creation of novel nucleic acid-based molecular devices capable of communication with specific proteases, regulated by their natural enzymatic activity. The overarching objective is to develop molecular tools that can convert protease-dependent cleavage of a peptide substrate into controlled signal processing and/or the release of biotherapeutics. These incorporate protease-responsive peptide nucleic acid (PNA)-peptide hybrids, which can be integrated into modular DNA-based systems for protease-dependent signal processing. This endeavor will yield new programmable molecular devices with the potential for applications in analytical chemistry, precision medicine, and synthetic biology.

2022 Excellent Science for Horizon Europe

Hybrid healthcare bionanomaterials actively controlled by biology

Role: Principal Investigator.

Funding body: Italian Ministry for University and Research and University of Parma.

 

The primary objective of this project is to pioneer the development of programmable nanotechnologies capable of precise activation, regulation, and in situ control by specific target biomolecules. Specifically, this project supports the creation of innovative biomedical technologies that can integrate sensing, processing, and response functionalities. These smart nanodevices leverage nanostructured materials in conjunction with synthetic biology components to execute customized chemical or biological functions in response to the recognition of specific biomolecules. These include nanoparticle formulations of nucleic acid nanomachines engineered for target-induced theragnostics, nanomaterials incorporating biological components into their bulk structure, enabling programmable bio-responsive properties, and nanomotors responsive to biomolecular inputs, facilitating controlled spatiotemporal manipulation within cellular environments.

This project plays a pivotal role in addressing critical challenges within the Health research and innovation domain of the PNR, aligning with the strategic plan “HealthTech for Society.” The activities undertaken in this project are central to the PNR Technologies for Health intervention area, focusing on the articulation “Bio-hybrid systems for new frontiers in biotechnology research and in precision and personalized medicine.” The aim of this project is to introduce programmable bioresponsive technologies that integrate an array of functionalities. In doing so, the sensing of specific biomolecules can be translated into chemical or biological actions with diagnostic or therapeutic implications.

FIL Giovani 2020 – Seed Grant for Young Researchers

Programmable chem-bio chimera translators as dynamic, functional probes for molecular sensing of informative protein markers

Role: Principal Investigator.

Funding body: University of Parma and CARIPARMA Foundation.

 

This project’s primary objective is to harness the expression of specific proteins, widely recognized as disease hallmarks, as potent indicators for screening, early diagnosis, and prognosis. Essential classes of proteins, including proteases, transcription factors, and other functional biomolecules, play pivotal roles in disease development and progression. In this context, protein-based molecular diagnostics play a fundamental role in enabling timely medical interventions. Therefore, there is an urgent need for strategies that enable sensitive, rapid, cost-effective, and unequivocal biomarker detection, particularly those capable of providing insights into the functional activity of the target protein.

Our approach capitalizes on the intrinsic ligand-binding and enzymatic activity inherent in specific marker proteins. We aim to utilize this functionality as the initial input of a sense-and-process detection system, enabling the engineering of protein-responsive molecular systems. In this innovative approach, the analyte of interest is not the protein itself, but rather its functional activity. DNA nanotechnology provides the basis for designing the proposed molecular devices. This project integrates elements of organic synthesis, analytical chemistry, and synthetic biology, synergistically combining these disciplines to create programmable biomolecular sensors specifically tailored for protein markers.