Alessandro Porchetta

• PhD
• PostDoc Position at University of Rome Tor Vergata

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units—a peptide and...

Here we report on the development of a CRISPR-based assay for the sensitive and specific detection of antibodies and antigens directly in complex sample matrices. The assay, called MAIGRET (Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription), is based on the use of a responsive synthetic DNA template that trigger the cell-f...

The RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in...

The RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in...

The detection of a protein analyte and use of this type of information for disease diagnosis and physiological monitoring requires methods with high sensitivity and specificity that have to be also easy to use, rapid and, ideally, single step. In the last 10 years, a number of DNA-based sensing methods and sensors have been developed in order to ac...

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units — a peptide an...

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units — a peptide an...

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks...

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross‐sectional geometry, cavity size, chirality, and length, while using only four DNA strand...

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but prog...

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross‐sectional geometry, cavity size, chirality, and length, while using only four DNA strand...

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Ca...

We report here the development of a cell-free in vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA...

Carbon nanotube (CNT)-based electrodes are cheap, highly performing, and robust platforms for the fabrication of electrochemical sensors. Engineering programmable DNA nanotechnologies on the CNT surface can support the construction of new electrochemical DNA sensors providing an amperometric output in response to biomolecular recognition. This is a...

We report here the development of a cell-free in-vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA...

We report here the development of a cell-free in-vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA...

The development of artificial vesicles into responsive architectures capable of sensing the biological environment and simultaneously signaling the presence of a specific target molecule is a key challenge in a range of biomedical applications from drug delivery to diagnostic tools. Herein, the rational design of biomimetic DNA‐grafted quatsome (QS...

Electrochemiluminescence (ECL) is a powerful transduction technique that has rapidly gained importance as a powerful analytical technique. Since ECL is a surface-confined process, a comprehensive understanding of the generation of ECL signal at a nanometric distance from the electrode could lead to several highly promising applications. In this wor...

We present a new class of DNA‐based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding‐upon‐repair DNA nanoswitches are synthetic DNA sequences containing O⁶‐methyl‐guanine (O⁶‐MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The na...

We present a new class of DNA‐based nanoswitches, named folding‐upon‐repair DNA nanoswitches, that upon enzymatic repair could undergo a conformational change mechanism leading to a change in fluorescent signal. The nanoswitches are suitable substrates for different methyltransferases and allow the screening of novel potential methyltransferase inh...

We demonstrate here a homogeneous assay, named NanoHybrid, for monoclonal antibody quantification directly in serum samples in a single step format. NanoHybrid is composed of both synthetic peptide nucleic acids (PNA) and nucleic acid strands conjugated to recognition elements and optical labels and is designed to allow a fast fluorescence quantifi...

Integrating dynamic DNA nanotechnology with protein‐controlled actuation will expand our ability to process molecular information. We have developed a strategy to actuate strand displacement reactions using DNA‐binding proteins by engineering synthetic DNA translators that convert specific protein‐binding events into trigger inputs through a progra...

The spatial colocalization and the nanoscale confinement of functional biomolecules in natural systems are widespread mechanisms that regulate a number of cell activities. This, alongside recent progresses in DNA nanotechnology, represents a source of inspiration for the design of artificial, nucleic acid‐based systems with programmable properties....

Using proteins to actuate DNA systems is challenging because it requires non‐trivial binding‐induced mechanisms. We demonstrate a strategy to perform DNA‐based molecular operations controlled by DNA‐binding proteins using synthetic DNA translators that convert specific protein‐binding events into trigger inputs through a programmable structural tra...

The fundamental concept of effective molarity is observed in a variety of biological processes, such as protein compartmentalization within organelles, membrane localization and signaling paths. To control molecular encountering and promote effective interactions, nature places biomolecules in specific sites inside the cell in order to generate a h...

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibo...

Integrating dynamic DNA nanotechnology with protein-controlled actuation will expand our ability to process molecular information. We have developed a strategy to actuate strand displacement reactions using DNA-binding proteins by engineering synthetic DNA translators that convert specific protein-binding events into trigger inputs through a progra...

Sensing in close quarters: A nucleic acid‐based electrochemical platform able to measure levels of immunoglobulins of type G and E (IgG and IgE) directly in blood serum and other bodily fluids was developed. The antibody detection exploits effective molarity effects that are induced by the spatial confinement of electrochemically active DNA‐based s...

In article number 1900023, Valeria Vetri, Bruno Pignataro, and co‐workers describe a novel inkjet‐printing approach that allows to downscale up to femtoliter‐scale water droplets in oil drops on a chip, triggering the generation of sub‐cellular size artificial compartments with self‐selected submicrometer‐thin molecular and biomolecular shell struc...

A simple, rapid, and highly controlled platform to prepare life‐inspired subcellular scale compartments by inkjet printing has been developed. These compartments consist of fL‐scale aqueous droplets (few µm in diameter) incorporating biologically relevant molecular entities with programmed composition and concentration. These droplets are ink‐jette...

The emerging field of RNA nanotechnology harnesses the versatility of RNA molecules to generate nature-inspired systems with programmable structure and functionality. Such methodology has therefore gained appeal in the fields of biosensing and diagnostics, where specific molecular recognition and advanced input/output processing are demanded. The u...

Bio-catalytic micro- and nanomotors have emerged as a new class of active matter self-propelled through enzymatic reactions. The incorporation of functional nanotools could enable the rational design of multifunctional micromotors for simultaneous real-time monitoring of their environment and activity. Herein, we report the combination of DNA nanot...

DNA nanotechnology employs synthetic nucleic acid strands to design and engineer nanoscale structural and functional systems of increasing complexity that may find applications in sensing,1-7 computing,8-10 molecular transport,11-13 information processing14 and catalysis.15,16 Several features make synthetic DNA a particularly appealing and advanta...

The increasing demand by citizens and environmental organization for the protection, preservation, and possible restoration of the marine environment has made seawater protection one of the urgent priorities of the European Union. In this contest, extensive monitoring and surveillance are required to correctly assess the current status of marine en...

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were o...

The detection of life markers is a high priority task in the exploration of the Solar System. Biochips performing in-situ multiplex immunoassays are a very promising approach alternative to gas chromatography coupled with mass spectrometry. As part of the PLEIADES project, we present the development of a chemiluminescence-based, highly integrated a...

DNA-based switches are structure-switching biomolecules widely employed in different bioanalytical applications. Of particular interest are DNA–based switches whose activity is regulated through the use of allostery. Allostery is a naturally occurring mechanism in which ligand binding induces the modulation and fine control of a connected biomolecu...

Antibody detection plays a pivotal role in the diagnosis of pathogens and monitoring the success of vaccine immunization. However, current serology techniques require multiple, time-consuming washing and incubation steps, which limit their applicability in point-of-care (POC) diagnostics and high-throughput assays. We developed here a nucleic acid...

One of the most intriguing ways through which Nature achieves regulation of biological pathways encompasses the coordination of non-covalent interactions that bring biomolecules to be co-localized in a designated restricted space. This nano-confinement leads to a drastic increase in the effective local concentration of the species involved, which u...

Here we demonstrate the rational design of a new class of DNA-based nanoswitches which are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nano...

Here we demonstrate a general and modular approach to regulate the activity of target-responsive DNA-based nanoswitches. We do so by coupling together two DNA-based responsive elements: a triplex-forming clamp-like probe able to bind a specific DNA sequence and a split aptamer selected to bind a small molecule. In the presence of the specific targe...

The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclus...

Here we demonstrate that we can rationally and finely control the functionality of different DNA-based nanodevices and nanoswitches using electronic inputs. To demonstrate the versatility of our approach we have used here three different model DNA-based nanoswitches triggered by heavy metals and specific DNA sequences and a copper-responsive DNAzym...

By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the Hybridization Chain Reaction (HCR), a pr...

Inspired by naturally occurring pH-regulated receptors, here we propose a rational approach to introduce pH-induced allostery into a wide range of DNA-based receptors. To demonstrate this we re-engineered two model DNA-based probes, a molecular beacon and a cocaine-binding aptamer, by introducing in their sequence a pH-dependent domain. We demonstr...

Here we explore a general strategy for the rational design of nucleic acid catalysts that can be allosterically activated by specific nucleic-acid binding proteins. To demonstrate this we have combined a catalytic DNAzyme sequence and the consensus sequence recognized by specific transcription factors to create a construct exhibiting two low-energy...

Achieving strategies to finely regulate with biological inputs the formation and functionality of DNA-based nanoarchitectures and nanomachines is essential towards a full realization of the potential of DNA nanotechnology. Here we demonstrate an unprecedented, rational approach to achieve control, through a simple change of the solution's pH, over...

Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by Cα-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was f...

Here we demonstrate the rational design of allosterically controllable, metal-ion-triggered molecular switches. Specifically, we designed DNA sequences that adopt two low energy conformations, one of which does not bind to the target ion and the other of which contains mismatches sites serving as specific recognition sites for mercury(II) or silver...

Here we demonstrate multiple, complementary approaches by which to tune, extend or narrow the dynamic range of aptamer-based sensors. Specifically, we have employed both distal site mutations and allosteric control to tune the affinity and dynamic range of a fluorescent aptamer beacon. We show that allosteric control, achieved by using a set of eas...

Mono and bi-component peptide-based self-assembled monolayers (SAMs) immobilized on a gold surface were studied by electrochemical and spectroscopic techniques. The peptides investigated were exclusively formed by Cα-tetrasubstituted amino acids. These residues, due to their peculiar conformational properties, constrain the peptide in a helical con...

The population-shift mechanism can be used for rational re-engineering of structure-switching biosensors to enable their allosteric inhibition and activation. As a proof-of-principle example of this, we have introduced distal allosteric sites into molecular beacons, which are optical sensors for the detection of specific nucleic acid sequences. The...

By combining DNA probes having different target affinities, but with similar specificity on the same electrode, an extended dynamic response of a biosensor spanning three orders of magnitude in target concentration was obtained. By using a different strategy, the useful dynamic range of an electrochemical DNA sensor was narrowed to only an 8-fold r...

A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8...

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified sing...

The photocurrent generation properties of mono- and bi-component peptide-based self-assembled monolayers (SAMs) immobilized on a gold surface were studied by electrochemical and spectroscopic techniques. The peptides investigated comprised almost exclusively C-tetrasubstituted -amino acids. These non-coded residues, because of their unique conforma...

Peptide foldamers based on alpha,alpha-disubstituted glycyl residues were synthesized and chemically characterized to investigate the effects of the electric field generated by a 3(10)-helix on the rate of intramolecular photoinduced electron-transfer reactions. To this end, two new octapeptides having identical sequences were suitably side-chain f...