Programming DNA-Based Systems through Effective Molarity Enforced by Biomolecular Confinement

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 high, localized concentration different from the bulk concentration. Inspired by this mechanism, scientists have artificially recreated in the lab the same strategy to actuate and control artificial DNA-based functional systems. Here, it is discussed how harnessing effective molarity has led to the development of a number of proximity-induced strategies, with applications ranging from DNA-templated organic chemistry and catalysis, to biosensing and protein-supported DNA assembly

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

DNA-based nanoswitches: insights into electrochemiluminescence signal enhancement

Electrochemiluminescence (ECL) is a powerful transduction technique that has rapidly gained importance as a powerful analytical technique. Since ECL is a surfaceconfined 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 work, we explored the mechanism underlying ECL signal generation on the nanoscale using luminophore-reporter-modified DNA-based nanoswitches (i.e., molecular beacon) with different stem stabilities. ECL is generated according to the "oxidative-reduction" strategy using tri-n-propylamine (TPrA) as a coreactant and Ru(bpy)(3)(2+) as a luminophore. Our findings suggest that by tuning the stem stability of DNA nanoswitches we can activate different ECL mechanisms (direct and remote) and, under specific conditions, a "digital-like" association curve, i.e., with an extremely steep transition after the addition of increasing concentrations of DNA target, a large signal variation, and low preliminary analytical performance (LOD 22 nM for 1GC DNA-nanoswtich and 16 nM for 5GC DNA-nanoswitch). In particular, we were able to achieve higher signal gain (i.e., 10 times) with respect to the standard "signal-off" electrochemical readout. We demonstrated the copresence of two different ECL generation mechanisms on the nanoscale that open the way for the design of customized DNA devices for highly efficient dual-signal-output ratiometric-like ECL systems

Challenges and perspectives of CRISPR-based technology for diagnostic applications

The precision and versatility of CRISPR-based techniques, combined with the advantages of nucleic acid-based nanotechnology, hold great promise in transforming the landscape of molecular diagnostics. While significant progress has been made, current CRISPR-based platforms primarly focus on nucleic acid detection. To expand the applicability and fully leverage the advantages offered by CRISPR-based diagnostics, ongoing efforts explore molecular strategies to develop CRISPR sensors capable of detecting a diverse range of analytes beyond nucleic acids. In addition, challenges still persist in the adaptation of CRISPR platforms for point-of-care (POC) applications, involving concerns such as portability and automation, as well as the complexities associated with multiplexing. Here, we provide a detailed classification and comprehensive discussion of molecular strategies facilitating the conversion of non-nucleic acid target binding into CRISPR-powered outputs with an emphasis on their corresponding design principles. Furthermore, the second part of the review outlines current challenges and potential solutions for seamlessly integrating these strategies into user-friendly platforms and rapid tests specifically tailored for point-of-care (POC)

Responsive Nucleic Acid-Based Organosilica Nanoparticles

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 progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol-gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing

Engineering DNA-grafted quatsomes as stable nucleic acid-responsive fluorescent nanovesicles

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) nanovesicles capable of translating the binding of a target molecule to amphiphilic DNA probes into an optical output is presented. QSs are synthetic lipid-based nanovesicles able to confine multiple organic dyes at the nanoscale, resulting in ultra-bright soft materials with attractiveness for sensing applications. Dye-loaded QS nanovesicles of different composition and surface charge are grafted with fluorescent amphiphilic nucleic acid-based probes to produce programmable FRET-active nanovesicles that operate as highly sensitive signal transducers. The photophysical properties of the DNA-grafted nanovesicles are characterized and the highly selective, ratiometric detection of clinically relevant microRNAs with sensitivity in the low nanomolar range are demonstrated. The potential applications of responsive QS nanovesicles for biosensing applications but also as functional nanodevices for targeted biomedical applications is envisaged

Supramolecular Nucleic Acid-Based Organosilica Nanoparticles Responsive to Physical and Biological Inputs

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 have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics

DNA-Based Scaffolds for Sensing Applications

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 advantageous biomaterial for all the above applications but more specifically for sensing. First, synthetic DNA sequences, especially if of limited length (<100 nucleotides), have highly predictable interactions and thermodynamics. This allows to develop spatio-temporally controlled nanostructures with quasi-Amstrong precision and to engineer supramolecular devices with well controlled secondary structures.17-22 DNA is also quite easy and inexpensive to synthetize: currently the cost of 150 µg of an unmodified single stranded DNA strand of 20 nucleotides is about 8 euros if purchased from one of the many commercial vendors available in the market. Finally, DNA is relatively stable if compared to other biomolecules like enzymes or antibodies. The other important feature of synthetic DNA is the wide range of possibilities that it offers for sensing applications if used as recognition element. Of course the most obvious use of a single stranded synthetic DNA sequence as recognition element is for the detection of a specific target complementary sequence. Countless applications of such use, especially if coupled with PCR, have been reported to date which resulted in many commercially available sensing devices.23,24 Synthetic DNA can also be used as recognition element for targets other than DNA. This is the case, for example, of DNA aptamers, a class of high-affinity nucleic acid ligands, which are selected through alternate cycles in vitro to bind a specific target molecule.25-29 To date, thousands of DNA and RNA aptamers have been selected which bind to specific targets including small molecules, proteins, peptides, bacteria, virus, and live cells.30-32 Other aptamers can bind to surface molecules and membrane proteins of live cells.33-35 A DNA aptamer is usually a short DNA sequence (<100 nucleotides) that can bind with high affinity (nM-µM) and high specificity its specific target. While the affinity of the aptamers is usually not as high as that of other biomolecular recognition elements (i.e. antibodies) there are some advantages connected with their use including the lower cost and the higher stability. Synthetic DNA can also be used as recognition element to detect metal ions through the use of thymine-thymine (T-T) and cytosine-cytosine (C-C) mismatches, which specifically bind mercury(II)36-38 and silver(I)39,40 ions respectively or through the use of copper-dependent DNAzymes.41 Similarly, the use of non-conventional DNA interactions can be used to rationally design pH-sensitive DNA switches that can be used as nanometer scale pH meters.42-44 Such probes typically exploit DNA secondary structures that display pH dependence due to the presence of specific protonation sites. These structures include I-motif,45-50 inter and intra molecular triplex DNA,51-55 DNA tweezers56 and catenanes.57 Recently, we have also reported on the rational design of programmable DNA-based nanoswitches whose closing/opening can be triggered over specific different pH windows by simply changing the relative content of TAT/CGC triplets in the switches.58 Finally, DNA can be employed as convenient recognition element for the detection of transcription factors, proteins that control the transcription of genetic information and that specifically recognize double-stranded or single-stranded DNA and RNA sequences.59-63

Allosterically regulated DNA-based switches: From design to bioanalytical applications

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 biomolecule function as a consequence of changes in concentration of the effector. Through this general mechanism, many different allosteric DNA-based switches able to respond in a highly controlled way at the presence of a specific molecular effector have been engineered. Here, we discuss how to design allosterically regulated DNA-based switches and their applications in the field of molecular sensing, diagnostic and drug release. (C) 2018 Published by Elsevier B.V