Nucleic Acid Scaffolds Research Articles

Nucleic Acid-Binding Dyes as Versatile Photocatalysts for Atom-Transfer Radical Polymerization.

Nucleic acid-binding dyes (NuABDs) are fluorogenic probes that light up after binding to nucleic acids. Taking advantage of their fluorogenicity, NuABDs have been widely utilized in the fields of nanotechnology and biotechnology for diagnostic and analytical applications. We demonstrate the potential of NuABDs together with an appropriate nucleic acid scaffold as an intriguing photocatalyst for precisely controlled atom-transfer radical polymerization (ATRP). Additionally, we systematically investigated the thermodynamic and electrochemical properties of the dyes, providing insights into the mechanism that drives the photopolymerization. The versatility of the NuABD-based platform was also demonstrated through successful polymerizations using several NuABDs in conjunction with diverse nucleic acid scaffolds, such as G-quadruplex DNA or DNA nanoflowers. This study not only extends the horizons of controlled photopolymerization but also broadens opportunities for nucleic acid-based materials and technologies, including nucleic acid-polymer biohybrids and stimuli-responsive ATRP platforms.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

Improving the Activity of Tryptophan Synthetase via a Nucleic Acid Scaffold.

Tryptophan synthetase (TSase), which functions as a tetramer, is a typical enzyme with a substrate channel effect, and shows excellent performance in the production of non-standard amino acids, histamine, and other biological derivatives. Based on previous work, we fused a mutant CE protein (colistin of E. coli, a polypeptide with antibacterial activity) sequence with the sequence of TSase to explore whether its catalytic activity could be enhanced, and we also analyzed whether the addition of a DNA scaffold was a feasible strategy. Here, dCE (CE protein without DNase activity) protein tags were constructed and fused to the TrapA and TrapB subunits of TSase, and the whole cell was used for the catalytic reaction. The results showed that after the dCE protein tag was fused to the TrapB subunit, its whole cell catalytic activity increased by 50%. Next, the two subunits were expressed separately, and the proteins were bound in vitro to ensure equimolar combination between the two subunits. After the dCE label was fused to TrapB, the activity of TSase assembled with TrapA also improved. A series of experiments revealed that the enzyme fused with dCE9 showed higher activity than the wild-type protein. In general, the activity of assembly TSase was optimal when the temperature was 50 °C and the pH was about 9.0. After a long temperature treatment, the enzyme maintained good activity. With the addition of exogenous nucleic acid, the activity of the enzyme increased. The maximum yield was 0.58 g/L, which was almost three times that of the wild-type TSase (0.21 g/L). The recombinant TSase constructed in this study with dCE fusion had the advantages of higher heat resistance and higher activity, and confirmed the feasibility of adding a nucleic acid scaffold, providing a new idea for the improvement of structurally similar enzymes.

Elucidating Sequence-Assembly Relationships for Bilingual PNA Biopolymers.

Nucleic acids and proteins possess encoded "languages" that can be used for information storage or to direct function. However, each biopolymer is limited to encoding its respective "language." Using a peptide nucleic acid (PNA) scaffold, nucleobase and amino acid residues can be installed on a singular backbone, enabling a single biopolymer to encode both languages. Our laboratory previously reported the development of a "bilingual" PNA biopolymer that incorporates a sequence-specific nucleic acid code interspersed with hydrophobic (alanine) and hydrophilic (lysine) amino acid residues at defined positions to produce amphiphilic character. We observed the amphiphilic amino acid residues directing the biopolymer to undergo self-assembly into micelle-like structures, while the nucleic acid recognition was harnessed for disassembly. Herein, we report a series of bilingual PNA sequences having amino acid residues with varying lengths, functional group charges, hydrophobicities, and spacings to elucidate the effect of these parameters on micelle assembly and nucleic acid recognition. Negative charges in the hydrophilic block or increased bulkiness of the hydrophobic side chains led to assembly into similarly sized micelles; however, the negative charge additionally led to increased critical micelle concentration. Upon PNA sequence truncation to decrease the spacing between side chains, the biopolymers remained capable of self-assembling but formed smaller structures. Characterization of disassembly revealed that each variant retained sequence recognition capabilities and stimuli-responsive disassembly. Together, these data show that the amino acid and nucleic acid sequences of amphiphilic bilingual biopolymers can be customized to finely tune the assembly and disassembly properties, which has implications for applications such as the encapsulation and delivery of cargo for therapeutics.

Oligonucleotides Containing C5-Propynyl Modified Arabinonucleic Acids: Synthesis, Biophysical and Antisense Properties.

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine (araUP ) and arabinocytidine (araCP ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araUP and araCP resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araUP and araCP in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

Activatable G-quadruplex based catalases for signal transduction in biosensing.

Discovery of oxidative catalysis with G-quadruplex•hemin constructs prompted a range of exciting developments in the field of biosensor design. Thus, G-quadruplex based DNAzymes with peroxidase activity found a niche as signal transduction modules in a wide range of analytical applications. The ability of nucleic acid scaffolds to recognise a variety of practically meaningful markers and to translate the recognition events into conformational changes powers numerous sensor design possibilities. In this work, we establish a catalase activity of G-quadruplex•hemin scaffolds. Catalase activated hydrogen peroxide decomposition generates molecular oxygen that forms bubbles. Observation of bubbles is a truly equipment free signal readout platform that is highly desirable in limited resources or do-it-yourself environments. We take a preliminary insight into a G-quadruplex structure-folding topology-catalase activity correlation and establish efficient operating conditions. Further, we demonstrate the platform's potential as a signal transduction modality for reporting on biomolecular recognition using an oligonucleotide as a proof-of-concept target. Ultimately, activatable catalases based on G-quadruplex•hemin scaffolds promise to become valuable contributors towards accessible molecular diagnostics applications.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that arefunctional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopystructures of thecomplexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer;have RNA threaded through the central channel of Rho;have 60 nucleotidesof RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotidesof RNA interacting sequence-specifically with the central channel of Rho;have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP;and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.

Ensemble Modified Aptamer Based Pattern Recognition for Adaptive Target Identification.

The difficulty of the molecular design and chemical synthesis of artificial sensing receptors restricts their diagnostic and proteomic applications. Herein, we report a concept of "ensemble modified aptamers" (EMAmers) that exploits the collective recognition abilities of a small set of protein-like side-chain-modified nucleic acid ligands for discriminative identification of molecular or cellular targets. Different types and numbers of hydrophobic functional groups were incorporated at designated positions on nucleic acid scaffolds to mimic amino acid side chains. We successfully assayed 18 EMAmer probes with differential binding affinities to seven proteins. We constructed an EMAmer-based chemical nose sensor and demonstrated its application in blinded unknown protein identification, giving a 92.9% accuracy. Additionally, the sensor is generalizable to the detection of blinded unknown bacterial and cellular samples, which enabled identification accuracies of 96.3% and 94.8%, respectively. This sensing platform offers a discriminative means for adaptive target identification and holds great potential for diverse applications.

Nucleic acid-based scaffold systems and application in enzyme cascade catalysis.

Enzymes, as elements with catalytic functions, can be rationally designed and multiple assembled to form a composite catalytic system and achieve cascade catalytic functions. Enzyme cascade catalysis could produce various chemical products with high conversion rate in short time. With the development of DNA nanotechnology, assembling enzymes to different nucleic acid-based scaffolds in different spatial organizations could effectively improve the catalytic efficiency of enzymes. Herein, we review the construction and application of nucleic acid-based scaffold systems from the perspective of template assembly in three dimensions. The challenges and future outlooks in the development of enzyme cascades are also discussed. KEY POINTS: • The principles and construction of various nucleic acid scaffolds are summarized • The application of nucleic acid scaffolds in enzyme cascade catalysis is discussed.

A multivalent activatable aptamer probe with ultralow background signal and high sensitivity for diagnosis of lung adenocarcinoma

Lung adenocarcinoma is the most common subtype of lung cancer. Developing a simple, rapid, and sensitive method for the detection of lung adenocarcinoma is helpful to reduce the clinical workload and facilitate clinical treatment. To detect lung adenocarcinoma cells, this study designed a multivalent activatable aptamer probe (multi-AP) using S1 aptamer that can specifically recognize A549 cells (a lung adenocarcinoma cell line). Firstly, the monovalent activatable aptamer probe (mono-AP) was designed by strand-displacement strategy and fluorescent resonance energy transfer (FRET). The multi-AP was constructed by mono-AP assembled on the nucleic acid scaffold obtained by hybridization chain reaction (HCR). The presence of the target cell activated the multi-AP, the complementary strand was released, and the fluorescence detection signal was reported based on the FRET. The multi-AP has good specificity and sensitivity for detecting A549 in different matrices and greatly reduces the background signal with the lowest detection limit of 6 cells/200 μL. Furthermore, the multi-AP could directly detect the lung adenocarcinoma cells in cytology specimens, indicating a good potential of the multi-AP in clinical cytology specimens for rapid diagnosis, such as rapid on-site cytological evaluation. Besides, the multi-AP was further modified with biotin (bio-multi-AP) and used to detect clinical cytology cell blocks, implying the value of bio-multi-AP in immunocytochemistry for accurate diagnosis of lung adenocarcinoma.

Modular nucleic acid scaffolds for synthesizing monodisperse and sequence-encoded antibody oligomers

Synthesizing protein oligomers that contain exact numbers of multiple different proteins in defined architectures is challenging. DNA-DNA interactions can be used to program protein assembly into oligomers; however, existing methods require changes to DNA design to achieve different numbers and oligomeric sequences of proteins. Herein, we develop a modular DNA scaffold that uses only six synthetic oligonucleotides to organize proteins into defined oligomers. As a proof-of-concept, model proteins (antibodies) are oligomerized into dimers and trimers, where antibody function is retained. Illustrating the modularity of this technique, dimer and trimer building blocks are then assembled into pentamers containing three different antibodies in an exact stoichiometry and oligomeric sequence. In sum, this report describes a generalizable method for organizing proteins into monodisperse, sequence-encoded oligomers using DNA. This advance will enable studies into how oligomeric protein sequences affect material properties in areas spanning pharmaceutical development, cascade catalysis, synthetic photosynthesis, and membrane transport.

Spt5‐nucleic acid interactions are directly involved in promoter proximal pausing of RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We use Drosophila melanogasteras a model system to address this question. We studied DSIF‐nucleic acid contacts made by the Spt5 NGN and KOW1 domains, which form a part of the upstream DNA exit tunnel, and the Spt5 KOW4 domain, which, along with the KOW5 domain, forms a clamp around the nascent RNA. Biochemical and structural studies have implicated these domains in pausing. We hypothesize that electrostatic interactions between Spt5 and the nucleic acid scaffold contribute to pausing by inhibiting the translocation of Pol II. We disrupted these electrostatic interactions by reversing the charge of key basic residues in Spt5 and used an electrophoretic mobility shift assay to measure Pol II binding and NELF recruitment. We then tested the pausing activity of the DSIF mutants in Drosophilanuclear extract depleted of wild‐type DSIF. Reversing the charge of six basic residues in the KOW1 domain resulted in weaker binding to Pol II. Though this mutant was still able to recruit NELF, it showed greatly reduced pausing activity. Reversing the charge of four basic residues in the KOW4 domain only modestly reduced binding to Pol II, had no impact on NELF recruitment, and significantly impaired DSIF’s pausing function. Thus, nucleic acid contacts made by the KOW1 domain of Spt5 promote pausing by mediating Pol II‐DSIF interactions (and therefore NELF recruitment) while the KOW4 domain interactions with the nascent transcript contribute to pausing directly. We also reversed the charge of two basic residues in the NGN domain and found that these changes had no effect on DSIF binding and NELF recruitment but did result in a modest decrease in pausing activity. One of these basic residues is an arginine located in an alpha helix sequence that is conserved in eukaryotes with NELF and promoter proximal pausing but is absent in eukaryotes lacking NELF. To test whether this alpha helix is required for promoter proximal pausing, we replaced the Drosophila helix sequence with unstructured loop sequences from Komagataella pastoris, Saccharomyces cerevisiae, and Caenorhabditis elegans, none of which have NELF. These mutants were able to bind Pol II and recruit NELF but exhibited significantly reduced pausing activity, suggesting that a short alpha helix in the NGN domain is critical for stabilizing the promoter proximal pause. This is likely achieved by optimally positioning a conserved arginine to interact with the DNA scaffold.

Arabinonucleic acids containing C5-propynyl modifications form stable hybrid duplexes with RNA that are efficiently degraded by E. coli RNase H

The promise of the antisense approach to treat a variety of diseases with oligonucleotides and solutions to challenges that have been encountered in their development is attributable to chemical modification of the nucleic acid scaffold. Herein, we describe preliminary data regarding the synthesis of a novel C5-propynyl-β-d-arabinouridine (araUP) phosphoramidite and its incorporation into oligonucleotides. Substitution of araUP in dT18 results in minor stabilization of duplexes formed with RNA when modifications are placed consecutively and a uniformly modified araUP 18-mer increases stability by 34 °C relative to DNA. The modified oligomer exhibits improved nuclease and serum stability when compared to DNA and duplexes formed between RNA and araUP oligonucleotides are substrates for E. coli RNase H. These preliminary results merit further investigation into C5-propynyl modified arabino nucleic acids for potential therapeutic gene silencing applications.

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

Several biological macromolecules adopt bivalent or multivalent interactions to perform various cellular processes. In this regard, the development of molecular constructs presenting multiple ligands in a specific manner is becoming crucial for the understanding of multivalent interactions and for the detection of target macromolecules. Nucleic acids are attractive molecules to achieve this goal because they are capable of forming various, structurally well-defined 2D or 3D nanostructures and can bear multiple ligands on their structures with precisely controlled ligand–ligand distances. Thanks to the features of nucleic acids, researchers have proposed a wide range of bivalent and multivalent binding agents that strongly bind to target biomolecules; consequently, these findings have uncovered new biosensing strategies for biomolecule detection. To date, various bivalent and multivalent interactions of nucleic acid architectures have been applied to the design of biosensors with enhanced sensitivity and target accuracy. In this review, we describe not only basic biosensor designs but also recently designed biosensors operating through the bivalent and multivalent recognition of nucleic acid scaffolds. Based on these designs, strategies to transduce bi- or multivalent interaction signals into readable signals are discussed in detail, and the future prospects and challenges of the field of multivalence-based biosensors are explored.

Signal Amplification Circuit Composed of Serinol Nucleic Acid for RNA Detection

Abstract A hybridization chain reaction (HCR) using a serinol nucleic acid (SNA) scaffold was newly designed and optimized. We found that hairpins with 8- or 9-mer loops, toeholds, and stems activated HCR and that the SNA interface accelerated initiation of HCR. Use of nitromethyl red as a quencher on the hairpin enabled detection of target RNA with high sensitivity. This system will be applicable to RNA detection in cell and biopsy, due to the high enzymatic durability of SNA.

Exosome-Encased Nucleic Acid Scaffold Chemotherapeutic Agents for Superior Anti-Tumor and Anti-Angiogenesis Activity.

Extracellular vesicles (EVs), or exosomes, play a pivotal role in tumor growth and metastasis, such as in the case of Kaposi Sarcoma. By loading tumor-derived EVs with chemotherapeutic drugs, we noted that their pro-tumor/pro-angiogenic phenotype was converted into an anti-tumor phenotype in vivo. Drug concentration in EVs was significantly higher than in clinically approved liposome formulation, as retention was facilitated by the presence of miRNAs inside the natural EVs. This demonstrates a new mechanism by which to increase the payload capacity of nanoparticles. By exploiting the targeting preferences of tumor-derived EVs, chemotherapeutics can be directed to specifically poison the cells and the microenvironment that enables metastasis.

Genetically Encodable Scaffolds for Optimizing Enzyme Function.

Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.

Dimeric G-Quadruplex: An Effective Nucleic Acid Scaffold for Lighting Up Thioflavin T.

As a recently identified higher-order quadruplex (G4) structure, the G4 dimer possesses unique structure and biological functions. In this work, we found accidentally that two tandem PW17 (one known G4-forming DNA) sequences can fold into a stable G4 dimer, and the G4 dimer can enhance dramatically the fluorescence intensity of thioflavin T (ThT). The G4 dimer/ThT fluorescence intensity is about ninefold that of the corresponding G4 monomer/ThT. Meanwhile, compared with the common G4/ThT system, G4 dimer/ThT exhibited more stable fluorescence emission in the media with various concentrations of Na+ and K+. On the basis of these findings, G4 dimer/ThT was used as a fluorescence indicator to construct one arched DNA probe for label-free detection of DNA. By incorporating a G4 dimer sequence in amplified products, we further designed one rolling circle amplification-based biosensing strategy to show the utility of this G4 dimer/ThT fluorescence indicator. This study demonstrates that dimeric G4 is an effective nucleic acid scaffold for lighting up ThT, showing promising applications in a label-free bioassay.