DNA-based Systems Research Articles

Predicting DNA Reactions with a Quantum Chemistry-Based Deep Learning Model.

In this study, a deep learning model based on quantum chemistry is introduced to enhance the accuracy and efficiency of predicting DNA reaction parameters. By integrating quantum chemical calculations with self-designed descriptor matrices, the model offers a comprehensive description of energy variations and considers a broad range of relevant factors. To overcome the challenge of limited labeled data, an active learning method is employed. The results demonstrate that this model outperforms existing methods in predicting DNA hybridization free energies and strand displacement rate constants, thus advancing the understanding of DNA molecular interactions, and aiding in the precise design and optimization of DNA-based systems.

Arginine-Modified Hemin Enhances G-Quadruplex DNAzyme Peroxidase Activity for High Sensitivity Detection.

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

Reconciling TD-DFT and CASPT2 electronic structure methods for describing the photophysics of DNA.

Time-dependent density functional theory (TD-DFT) and multiconfigurational second-order perturbation theory (CASPT2) are two of the most widely used methods to investigate photoinduced dynamics in DNA-based systems. These methods sometimes give diverse dynamics in physiological environments usually modeled by quantum mechanics/molecular mechanics (QM/MM) protocol. In this work, we demonstrate for the uridine test case that the underlying topology of the potential energy surfaces of electronic states involved in photoinduced relaxation is similar in both electronic structure methods. This is verified by analyzing surface-hopping dynamics performed at the QM/MM level on aqueous solvated uridine at TD-DFT and CASPT2 levels. By constraining the dynamics to remain on state we observe similar fluctuations in energy and relaxation lifetimes in surface-hopping dynamics in both TD-DFT and experimentally validated CASPT2 methods. This finding calls for a systematic comparison of the ES potential energy surfaces of DNA and RNA nucleosides at the single- and multi-reference levels of theory. The anomalous long excited state lifetime at the TD-DFT level is explained by trapping due to the tendency of TD-DFT in QM/MM schemes with electrostatic embedding to underestimate the energy of the state leading to a wrong energetic order. A study of the FC energetics suggests that improving the description of the surrounding environment through polarizable embedding or by the expansion of QM layer with hydrogen-bonded waters helps restore the correct state order at TD-DFT level. Thus by combining TDDFT with an accurate modeling of the environment, TD-DFT is positioned as the standout protocol to model photoinduced dynamics in DNA-based aggregates and multimers.

Enhancing DNA-based nanodevices activation through cationic peptide acceleration of strand displacement.

Dynamic DNA-based nanodevices offer versatile molecular-level operations, but the majority of them suffer from sluggish kinetics, impeding the advancement of device complexity. In this work, we present the self-assembly of a cationic peptide with DNA to expedite toehold-mediated DNA strand displacement (TMSD) reactions, a fundamental mechanism enabling the dynamic control and actuation of DNA nanostructures. The target DNA is modified with a fluorophore and a quencher, so that the TMSD process can be monitored by recording the time-dependent fluorescence changes. The boosting effect of the peptides is found to be dependent on the peptide/DNA N/P ratio, the toehold/invader binding affinity, and the ionic strength with stronger effects observed at lower ionic strengths, suggesting that electrostatic interactions play a key role. Furthermore, we demonstrate that the cationic peptide enhances the responsiveness and robustness of DNA machinery tweezers or logic circuits (AND and OR) involving multiple strand displacement reactions in parallel and cascade, highlighting its broad utility across DNA-based systems of varying complexity. This work offers a versatile approach to enhance the efficiency of toehold-mediated DNA nanodevices, facilitating flexible design and broader applications.

Self-Assembly of Designed Peptides with DNA to Accelerate the DNA Strand Displacement Process for Dynamic Regulation of DNAzymes.

Toehold-mediated DNA strand displacement (TMSD) is a powerful tool for controlling DNA-based molecular reactions and devices. However, the slow kinetics of TMSD reactions often limit their efficiency and practical applications. Inspired by the chemical structures of natural DNA-operating enzymes (e.g., helicase), we designed lysine-rich peptides to self-assemble with DNA-based systems. Our approach allows for accelerating the TMSD reactions, even during multiple displacement events, enhancing their overall efficiency and utility. We found that the acceleration is dependent on the peptide's sequence, length, and concentration as well as the length of the DNA toehold domain. Molecular dynamics simulations revealed that the peptides promote toehold binding between the double-stranded target and the single-stranded invader, thereby facilitating strand displacement. Furthermore, we integrated our approach into a horseradish peroxidase-mimicking DNAzyme, enabling the dynamic modulation of enzymatic functions on and off. We anticipate that the established acceleration of strand displacement reactions and the modulation of enzymatic activities offer enhanced functionality and control in the design of programmable DNA-based nanodevices.

Enzyme-Linked DNA Displacement (ELIDIS) Assay for Ultrasensitive Electrochemical Detection of Antibodies.

Here we report the development of a method for the electrochemical ultrasensitive detection of antibodies that couples the programmability and versatility of DNA-based systems with the sensitivity provided by enzymatic amplification. The platform, termed Enzyme-Linked DNA Displacement (ELIDIS), is based on the use of antigen-DNA conjugates that, upon the bivalent binding of a specific target antibody, induce the release of an enzyme-DNA hybrid strand from a preformed duplex. Such enzyme-DNA hybrid strand can then be electrochemically detected with a disposable electrode with high sensitivity. We applied ELIDIS to demonstrate the sensitive (limit of detection in the picomolar range), specific and multiplexed detection of five different antibodies including three clinically relevant ones. ELIDIS is also rapid (it only requires two reaction steps), works well in complex media (serum) and is cost-effective. A direct comparison with a commercial ELISA kit for the detection of Cetuximab demonstrates the promising features of ELIDIS as a point-of-care platform for antibodies detection.

The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection.

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed VHHs and the antigen present in the cell, preventing the necessity to remove the excess unbound VHHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

Development of Synthetic mRNAs Encoding Split Cytotoxic Proteins for Selective Cell Elimination Based on Specific Protein Detection.

For the selective elimination of deleterious cells (e.g., cancer cells and virus-infected cells), the use of a cytotoxic gene is a promising approach. DNA-based systems have achieved selective cell elimination but risk insertional mutagenesis. Here, we developed a synthetic mRNA-based system to selectively eliminate cells expressing a specific target protein. The synthetic mRNAs used in the system are designed to express an engineered protein pair that are based on a cytotoxic protein, Barnase. Each engineered protein is composed of an N- or C-terminal fragment of Barnase, a target protein binding domain, and an intein that aids in reconstituting full-length Barnase from the two fragments. When the mRNAs are transfected to cells expressing the target protein, both N- and C-terminal Barnase fragments bind to the target protein, causing the intein to excise itself and reconstitute cytotoxic full-length Barnase. In contrast, when the target protein is not present, the reconstitution of full-length Barnase is not induced. Four candidate constructs containing split Barnase were evaluated for the ability to selectively eliminate target protein-expressing cells. One of the candidate sets demonstrated highly selective cell death. This system will be a useful therapeutic tool to selectively eliminate deleterious cells.

Dissipative DNA nanotechnology.

DNA nanotechnology has emerged as a powerful tool to precisely design and control molecular circuits, machines and nanostructures. A major goal in this field is to build devices with life-like properties, such as directional motion, transport, communication and adaptation. Here we provide an overview of the nascent field of dissipative DNA nanotechnology, which aims at developing life-like systems by combining programmable nucleic-acid reactions with energy-dissipating processes. We first delineate the notions, terminology and characteristic features of dissipative DNA-based systems and then we survey DNA-based circuits, devices and materials whose functions are controlled by chemical fuels. We emphasize how energy consumption enables these systems to perform work and cyclical tasks, in contrast with DNA devices that operate without dissipative processes. The ability to take advantage of chemical fuel molecules brings dissipative DNA systems closer to the active molecular devices that exist in nature.

Programmable Cell-Free Transcriptional Switches for Antibody Detection.

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 aptamer. The system couples the unique programmability and responsiveness of DNA-based systems with the specificity and sensitivity offered by in vitro genetic circuitries and commercially available transcription kits. We demonstrate that cell-free transcriptional switches can efficiently measure antibody levels directly in blood serum. Thanks to the programmable nature of the sensing platform, the method can be adapted to different antibodies: we demonstrate here the sensitive, rapid, and cost-effective detection of three different antibodies and the possible use of this approach for the simultaneous detection of two antibodies in the same solution.

Horticultural innovation by viral-induced gene regulation of carotenogenesis.

Multipartite viral vectors provide a simple, inexpensive and effective biotechnological tool to transiently manipulate (i.e. reduce or increase) gene expression in planta and characterise the function of genetic traits. The development of virus-induced gene regulation (VIGR) systems usually involve the targeted silencing or overexpression of genes involved in pigment biosynthesis or degradation in plastids, thereby providing rapid visual assessment of success in establishing RNA- or DNA-based VIGR systems in planta. Carotenoids pigments provide plant tissues with an array of yellow, orange, and pinkish-red colours. VIGR-induced transient manipulation of carotenoid-related gene expression has advanced our understanding of carotenoid biosynthesis, regulation, accumulation and degradation, as well as plastid signalling processes. In this review, we describe mechanisms of VIGR, the importance of carotenoids as visual markers of technology development, and knowledge gained through manipulating carotenogenesis in model plants as well as horticultural crops not always amenable to transgenic approaches. We outline how VIGR can be utilised in plants to fast-track the characterisation of gene function(s), accelerate fruit tree breeding programs, edit genomes, and biofortify plant products enriched in carotenoid micronutrients for horticultural innovation.

Using Threshold DNA Strand Displacement to Construct a Half Adder

DNA strand displacement has the advantages of product predictability, programmability and threshold, and it is often used to build DNA-based logic operation systems. In this paper, we use DNA strand displacement to have different reaction priorities in different length ranges of the toehold domain to form the effect of the threshold and construct the logical AND gate and XOR gate. Logical operations use single-stranded DNA as the input signal, and the brightness of the fluorescence is used to measure the results. Then, the logic AND gate and XOR gate are used as basic logic units to form a half adder in parallel. Finally, we use Visual DSD to simulate and analyze the logic AND gate, XOR gate and half adder. The simulation results show that the logic gates constructed in this paper have good theoretical feasibility and effectiveness. This work provides a potential design idea for DNA-based arithmetic function operations and more advanced logic operations.

Venturing into auditing of reference libraries: from the hackathon on marine invertebrates to sorting with BAGS

Reference libraries of DNA sequences are the backbone of DNA-based taxonomic identification systems. The quality and accuracy of the data in reference libraries is critical to achieve reliable identifications. Faulty or inaccurate data may have detrimental impacts in various downstream applications, perpetuating errors over long-term studies and biodiversity data repositories. This risk is particularly prevalent in metabarcoding approaches, where millions of sequences are assigned to taxa in reference libraries through automated and frequently unsupervised procedures. Although quality-compliance measures have been implemented in several stages of the DNA barcode production workflow, no systematized approach has tackled the challenges of revision, curation and annotation of reference libraries. The trend for increasing detection of cryptic diversity further complicates this task. Here we outline the conclusions of the application of two distinct approaches to audit and annotate reference libraries: the hackathon on marine invertebrates hosted by the 8th IBOL conference, and the bioinformatics application “Barcode, Audit & Grade System” (BAGS; Fontes et al. 2021). The former consisted on the assembly of 18 researchers involved in marine barcoding, aiming to audit and annotate a very large number DNA barcode records available in BOLD from major marine invertebrate taxa, including all or selected groups of Annelida, Crustacea, Echinodermata and Mollusca. Discordant Barcode Index Numbers (BINs), that is, BINs including more than one species, were reviewed individually, and the respective records annotated with one of the 4 following tags: MIS-ID (misidentification); AMBIG (ambiguous, unable to resolve); COMPLEX (multiple BINs); SHARE (barcodes shared among species in the same BIN). This effort resulted in the processing of >80.000 barcodes, corresponding to >7.500 species, of which 7% were tagged MIS-ID, 17% AMBIG, 13% COMPLEX and 1% SHARE, with Gastropoda displaying particularly high levels of ambiguity. The sizeable portion of MIS-ID and AMBIG tags raises concern. Yet, part of the AMBIG tags merely reflect underlying uncertainty in species taxonomic status, rather than the deposition of erroneous data in BOLD. Hence, in addition to auditing and annotation, extensive effort should continue to be allocated to the underpinning alpha taxonomy of reference libraries. The second approach here described is BAGS, which consists on an R-based application that provides an user-friendly platform for automated auditing of user-selected metazoan cytochrome oxidase I (COI) reference libraries. BAGS sorts BOLD’s records and species into 5 grades, depending on whether they display BIN concordance (A, B) multiple BINs (C), less than two records (D) or discordant BINs (E). A WoRMS-linked filter allows to select or exclude marine taxa, and a reporting component provides a graphical overview and FASTA files assorted in different combinations of grades. Therefore, BAGS can provide a quick appraisal of the status of an user-defined reference library, allowing simultaneously to recognize the most reliable records, the incidence of cases high intraspecific divergence, gaps in representativeness, and inaccuracies of potential concern. A pilot assessment of BAGS performance in three datasets comprising marine fish, Chironomidae (Insecta) and marine Amphipoda (Crustacea) highlighted the differences in the congruence status of the respective reference libraries. In conclusion, the hackathon had and expressive contribution to the revision and annotation of a very large number of marine invertebrate records lodged in BOLD. Human-mediated revision is highly-reliable and consequential, however, it constituted a massive undertaking that can hardly be repeated without a previous refinement and substantial reduction of the datasets to be revised. This could be achieved resorting to automated revision systems, among which BAGS constitutes a first step. We intend to progress with the expansion and improvement of BAGS, namely by introducing further refinements in the analyses of grade E data, in order to automatically discard simple cases of discordance, thereby reducing the amount of data needing human-mediated revision. Recognition of the need for automated reference library auditing and curation systems is essential to raise confidence of researchers, environmental managers and governmental agencies for the adoption and implementation of DNA-based approaches in aquatic biomonitoring.

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.

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

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

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes

The selective transport of ions across cell membranes, controlled by membrane proteins, is critical for a living organism. DNA-based systems have emerged as promising artificial ion transporters. However, the development of stable and selective artificial ion transporters remains a formidable task. We herein delineate the construction of an artificial ionophore using a telomeric DNA G-quadruplex (h-TELO) and a lipophilic guanosine (MG). MG stabilizes h-TELO by non-covalent interactions and, along with the lipophilic side chain, promotes the insertion of h-TELO within the hydrophobic lipid membrane. Fluorescence assays, electrophysiology measurements and molecular dynamics simulations reveal that MG/h-TELO preferentially transports K+-ions in a stimuli-responsive manner. The preferential K+-ion transport is presumably due to conformational changes of the ionophore in response to different ions. Moreover, the ionophore transports K+-ions across CHO and K-562 cell membranes. This study may serve as a design principle to generate selective DNA-based artificial transporters for therapeutic applications.

Molecular access to multi-dimensionally encoded information

Polymer scientist have only recently realized that information storage on the molecular level is not only restricted to DNA-based systems. Similar encoding and decoding of data have been demonstrated on synthetic polymers that could overcome some of the drawbacks associated with DNA, such as the ability to make use of a larger monomer alphabet. This feature article describes some of the recent data storage strategies that were investigated, ranging from writing information on linear sequence‑defined macromolecules up to layer-by-layer casted surfaces and QR codes. In addition, some strategies to increase storage density are elaborated and some trends regarding future perspectives on molecular data storage from the literature are critically evaluated. This work ends with highlighting the demand for new strategies setting up reliable solutions for future data management technologies.

Opportunities and Challenges in DNA-Hybrid Nanomaterials.

Nature has inspired the development of many life-like materials. Although still simplistic, key biological functionalities have been incorporated, enabling a wide variety of applications. DNA-based systems, in particular, show high promise due to their ability to merge specific Watson-Crick base pairing with unique properties that are also programmable, scalable, or dynamic. By combining the fields of DNA-based covalent polymers, DNA origami, and DNA-functionalized supramolecular polymers, new frontiers in next-generation DNA-based hybrid materials that can outperform current bioartificial systems will be realized. Many challenges must still be overcome before this emerging technology can be materialized.

Novel enzymatic single-nucleotide modification of DNA oligomer: prevention of incessant incorporation of nucleotidyl transferase by ribonucleotide-borate complex.

Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide–borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The ‘TdT in rNTP-borate’ method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.