Alessandro Porchetta

Using Distal-Site Mutations and Allosteric Inhibition To Tune, Extend, and Narrow the Useful Dynamic Range of Aptamer-Based Sensors

Here we demonstrate multiple, complementary approaches by which to tune, extend, or narrow the dynamic range of aptamer-based sensors. Specifically, we employ 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 easily designed oligonucleotide inhibitors that competes against the folding of the aptamer, allows rational fine-tuning of the affinity of our model aptamer across 3 orders of magnitude of target concentration with greater precision than that achieved using mutational approaches. Using these methods, we generate sets of aptamers varying significantly in target affinity and then combine them to recreate several of the mechanisms employed by nature to narrow or broaden the dynamic range of biological receptors. Such ability to finely control the affinity and dynamic range of aptamers may find many applications in synthetic biology, drug delivery, and targeted therapies, fields in which aptamers are of rapidly growing importance.

Allosterically Tunable, DNA-Based Switches Triggered by Heavy Metals

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 mismatch sites serving as specific recognition elements for mercury(II) or silver(I) ions. Both switches contain multiple metal binding sites and thus exhibit homotropic allosteric (cooperative) responses. As heterotropic allosteric effectors we employ single-stranded DNA sequences that either stabilize or destabilize the nonbinding state, enabling dynamic range tuning over several orders of magnitude. The ability to rationally introduce these effects into target-responsive switches could be of value in improving the functionality of DNA-based nanomachines.

Re‐engineering Electrochemical Biosensors To Narrow or Extend Their Useful Dynamic Range

Here we demonstrate two convenient methods to extend and narrow the useful dynamic range of a model electrochemical DNA sensor. We did so by combining DNA probes of different target affinities but with similar specificity on the same electrode. We were able to achieve an extended dynamic response spanning 3 orders of magnitude in target concentration. Using a different strategy we have also narrowed the useful dynamic range of an E-DNA sensor to only an 8-fold range of target concentrations.

Rational Design of pH-Controlled DNA Strand Displacement

Achieving strategies to finely regulate with biological inputs the formation and functionality of DNA-based nanoarchitectures and nanomachines is essential toward 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 solutions pH, over an important class of DNA association-based reactions. To do so we took advantage of the pH dependence of parallel Hoogsteen interactions and rationally designed two triplex-based DNA strand displacement strategies that can be triggered and finely regulated at either basic or acidic pHs. Because pH change represents an important input both in healthy and pathological biological pathways, our findings can have implication for the development of DNA nanostructures whose assembly and functionality can be triggered in the presence of specific biological targets.

General Strategy to Introduce pH-Induced Allostery in DNA-Based Receptors to Achieve Controlled Release of Ligands.

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 demonstrate here that we can finely tune the affinity of these model receptors and control the load/release of their specific target molecule by a simple pH change.

Probe accessibility effects on the performance of electrochemical biosensors employing DNA monolayers

AbstractSurface-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 single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets. FigureHere we demonstrate that the density of the DNA probe monolayer is a crucial determinant of the final signalling of DNA-bases sensors used for the detection of proteins, enzymes, and other macromolecules.

Controlling Hybridization Chain Reactions with pH

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 process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures.

Photocurrent generation through peptide‐based self‐assembled monolayers on a gold surface: antenna and junction effects

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 Cα‐tetrasubstituted α‐amino acids. These non‐coded residues, because of their unique conformational properties, forced the peptide backbone to attain a helical conformation, as confirmed by X‐ray crystal structure and CD determinations in solution. The peptide helical structure promoted the formation of a stable SAM on the gold surface, characterized by an electric macrodipole directed from the C(δ−) to the N(δ+) terminus, that remarkably affected the electron transfer (ET) process through the peptide chain. The peptides investigated were derivatized with chromophores strongly absorbing in the UV region to enhance the efficiency of the photocurrent generation (antenna effect). The influence of the nature of the peptide–gold interface on the ET process (junction effect) was analyzed by comparing the photocurrent generation process in peptide SAMs immobilized on a gold surface through AuS linkages with that in a bi‐component SAM embedding a photoactive peptide into the linked palisade formed by disulfide‐functionalized peptides. Copyright

Conformational effects on the electron-transfer efficiency in peptide foldamers based on alpha,alpha-disubstituted glycyl residues.

Peptide foldamers based on α,α‐disubstituted glycyl residues were synthesized and chemically characterized to investigate the effects of the electric field generated by a 310‐helix on the rate of intramolecular photoinduced electron‐transfer reactions. To this end, two new octapeptides having identical sequences were suitably side‐chain functionalized with the same electron‐transfer donor–acceptor pair, but inverting the position of the pair along the main chain. The electron‐transfer rate constants, measured by time‐resolved spectroscopy techniques (nanosecond transient absorption and time‐resolved fluorescence), indicated that, in the case of the 310‐helix, the electrostatic effect is significant, but smaller than that obtained for α‐helical peptides. This finding can be likely ascribed to the distortion of the H‐bond network with respect to the helical axis taking place in the former secondary structure. Overall, these results could have implications on electron‐transfer phenomena in model and biomembranes facilitated by peptaibiotics.

Photoinduced Electron Transfer through Peptide-Based Self-Assembled Monolayers Chemisorbed on Gold Electrodes: Directing the Flow-in and Flow-out of Electrons through Peptide Helices

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 C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.