Design of specific nucleic acid‐based biosensors for protein binding activity

Nucleic acid-based biosensors for the detection of specific proteins combine the typical programmability of synthetic DNA systems with artificially controlled DNA-protein communication. The high-affinity interaction between a target protein and a specific ligand, such as an aptamer sequence, or a double stranded DNA domain, or a small peptide, is paired with a nature-mimicking molecular mechanism allowing for probing, processing, and translating protein binding activity into a measurable signal. In this Review, two main strategies developed in the context of protein-responsive nucleic acid-based biosensors are discussed. One is the design of proximity-based assays harnessing the spatial colocalization of functional probes within the volume of a multivalent protein. The other is the engineering of dynamic DNA structures that undergo a controlled conformational or structural change upon protein binding. Examples of applications from optical and electrochemical detection of antibodies in biofluids to fluorescence imaging of transcription factors in living cells are reported, and suggestions along with possible future directions in the field are discussed

Using antibodies to control DNA-templated chemical reactions

DNA-templated synthesis takes advantage of the programmability of DNA-DNA interactions to accelerate chemical reactions under diluted conditions upon sequence-specific hybridization. While this strategy has proven advantageous for a variety of applications, including sensing and drug discovery, it has been so far limited to the use of nucleic acids as templating elements. Here, we report the rational design of DNA templated synthesis controlled by specific IgG antibodies. Our approach is based on the co-localization of reactants induced by the bivalent binding of a specific IgG antibody to two antigen-conjugated DNA templating strands that triggers a chemical reaction that would be otherwise too slow under diluted conditions. This strategy is versatile, orthogonal and adaptable to different IgG antibodies and can be employed to achieve the targeted synthesis of clinically-relevant molecules in the presence of specific IgG biomarker antibodies

Engineering DNA-Grafted Quatsomes as Stable Nucleic Acid-Responsive Fluorescent Nanovesicles

Fluorescence; Nanovesicles; Responsive nanomaterialsFluorescencia; Nanovesículas; Nanomateriales sensiblesFluorescència; Nanovesícules; Nanomaterials sensiblesThe 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.This work was financially supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement “Nano-Oligo Med” (No 778133), Ministry of Science and Innovation (MINECO), Spain, through the “MOL4BIO” project (PID2019-105622RB-I00) and by Instituto de Salud Carlos III (DTS20/00018), Italian Ministry of University and Research (Project of National Interest, PRIN, 2017Y2PAB8_004 through the project “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”. M.R. was supported from a Fondazione Umberto Veronesi postdoctoral fellowship. Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centers of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Quatsome production and their physicochemical characterization has been performed by the Biomaterial Processing and Nanostructuring Unit (U6) of the ICTS “NANBIOSIS”, a unit of the CIBER network in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC)

Bispecific antibody detection using antigen-conjugated synthetic nucleic acid strands

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches

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.This work was financially supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska- Curie grant agreement “Nano-Oligo Med” (No 778133), Ministry of Science and Innovation (MINECO), Spain, through the “MOL4BIO” project (PID2019-105622RB-I00) and by Instituto de Salud Carlos III (DTS20/00018), Italian Ministry of University and Research (Project of National Interest, PRIN, 2017Y2PAB8_004 through the project “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”. M.R. was supported from a Fondazione Umberto Veronesi postdoctoral fellowship. Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centers of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Quatsome production and their physicochemical characterization has been performed by the Biomaterial Processing and Nanostructuring Unit (U6) of the ICTS “NANBIOSIS”, a unit of the CIBER network in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC).Peer reviewe

Synthetic DNA-based nanoswimmers driven by enzyme catalysis

We report here DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA enzyme nanoswimmers) that self-propel by converting the enzymatic substrate into the product in solution. The DNA enzyme nanoswimmers are obtained from tubular DNA structures that self-assemble spontaneously by hybridization of DNA tiles. We have functionalized these DNA structures with two different enzymes, urease and catalase, and show that upon addition of the enzymatic substrate (i.e., urea and H2O2), they exhibit concentration-dependent motion and different motion dynamics, including enhanced diffusion and ballistic motion. These results pave the way for the development of synthetic enzyme-driven nanoswimmers that can self-propel in fluids and have the potential to provide new insights into biological motion dynamics at the micro-nanoscale

Filamin-A regulates actin-dependent clustering of HIV receptors

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infectio