Single-stranded Domain Research Articles

Mismatch-Guided Deoxyribonucleic Acid Assembly Enables Ultrasensitive and Multiplex Detection of Low-Allele-Fraction Variants in Clinical Samples.

Somatic mutations are important signatures in clinical cancer treatment. However, accurate detection of rare somatic mutations with low variant-allele frequencies (VAFs) in clinical samples is challenging because of the interference caused by high concentrations of wild-type (WT) sequences. Here, we report a post amplification SNV-specific DNA assembly (PANDA) technology that eliminates the high concentration pressure caused by WT through a mismatch-guided DNA assembly and enables the ultrasensitive detection of cancer mutations with VAFs as low as 0.1%. Because it generates an assembly product that only exposes a single-stranded domain with the minimal length for signal readout and thus eliminates possible interferences from secondary structures and cross-interactions among sequences, PANDA is highly versatile and expandable for multiplex testing. With ultrahigh sensitivity, PANDA enabled the quantitative analysis of EGFR mutations in cell-free DNA of 68 clinical plasma samples and four pleuroperitoneal fluid samples, with test results highly consistent with NGS deep sequencing. Compared to digital PCR, PANDA returned fewer false negatives and ambiguous cases of clinical tests. Meanwhile, it also offers much lower upfront instrumental and operational costs. The multiplexity was demonstrated by developing a 3-plex PANDA for the simultaneous analysis of three EGFR mutations in 54 pairs of tumor and the adjacent noncancerous tissue samples collected from lung cancer patients. Because of the ultrahigh sensitivity, multiplexity, and simplicity, we anticipate that PANDA will find wide applications for analyzing clinically important rare mutations in diverse devastating diseases.

Kineto-Mechanistic Investigation of Effect of Macromolecular Crowding on the Breathing of DNA Bubble.

Macromolecular crowding along with hydrogen bonding or stacking interactions and hydration reportedly has enormous repercussions on elementary biochemical processes, such as the folding of proteins or nucleic acids involving the stability of DNA base pairing. By using the mismatch-induced DNA bubble as a mesoscopic model, the complex interplay of macromolecular crowding on the dynamical fluctuations at the bubble region within the thermodynamic limit has been monitored using single-molecule fluorescence resonance energy transfer (sm-FRET). These single-molecule experimental results have been further corroborated using physical models such as "scaled particle theory" (SPT) and "Gaussian cloud model" (GCM), to predict the biological activity of DNA. The two-state fluctuation of the DNA bubble has been visualized as a function of the nature, size, and concentration of the crowder. The influence of crowders on the DNA conformation has been investigated with the help of the m-factor, the eccentricity, and the kinetic and thermodynamic parameters without any prior assumption. The clear effect of crowding on the dynamics of such a simple biomolecular system emphasizes the power of single-molecule methods and the dependency of the radius of gyration of the co-solute as well as the preferential interaction with the crowder on the distinct conformational states adopted by the bubble. This study provides an idea and hypothesizes the preferential propensity of the DNA bubble to adopt a conformation with the single-stranded domains being far apart, independent of the crowder size, that may be beneficial for efficient recognition by proteins for an uninterrupted procession of the biological process of the central dogma.

Computational Design of RNA Toehold-Mediated Translation Activators.

Translation activators are an important class of riboregulators that respond to nucleic acid signals by activating gene expression. Toehold switches and single-nucleotide-specific programmable riboregulators (SNIPRs) are two types of translation activators that can detect nearly any nucleic acid sequence using interactions initiated by single-stranded domains known as toeholds. Toehold switches operate with high dynamic range, orthogonality, and programmability, making them capable of detecting a variety of pathogens in paper-based cell-free diagnostic assays. SNIPRs are designed to enable the accurate detection of single-nucleotide mutations, making them valuable tools for mutation and drug-resistance assays. Here we describe the computational design process for generating toehold switches and SNIPRs active against different pathogens and mutations of interest. Such riboregulators can be deployed in paper-based diagnostic assays to enable rapid and low-cost disease detection.

Dynamic and scalable DNA-based information storage

The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.

Context-Sensitive Cleavage of Folded DNAs by Loop-Targeting bPNAs.

Herein, we demonstrate context-dependent molecular recognition of DNA by synthetic bPNA iron and copper complexes, using oxidative backbone cleavage as a chemical readout for binding. Oligoethylenimine bPNAs displaying iron·EDTA or copper·phenanthroline sites were found to be efficient chemical nucleases for designed and native structured DNAs with T-rich single-stranded domains. Cleavage reactivity depends strongly on structural context, as strikingly demonstrated with DNA substrates of the form (GGGTTA)n. This repeat sequence from the human telomere is known to switch between parallel and antiparallel G-quadruplex (G4) topologies with a change from potassium to sodium buffer: notably, bPNA-copper complexes efficiently cleave long repeat sequences into ∼22-nucleotide portions in sodium, but not potassium, buffer. We hypothesize preferential cleavage of the antiparallel topology (Na+) over the parallel topology (K+) due to the greater accessibility of the TTA loop to bPNA in the antiparallel (Na+) form. Similar ion-sensitive telomere shortening upon treatment with bPNA nucleases can be observed in both isolated and intracellular DNA from PC3 cells by quantitative polymerase chain reaction. Live cell treatment was accompanied by accelerated cellular senescence, as expected for significant telomere shortening. Taken together, the loop-targeting approach of bPNA chemical nucleases complements prior intercalation strategies targeting duplex and quadruplex DNA. Structurally sensitive loop targeting enables discrimination between similar target sequences, thus expanding bPNA targeting beyond simple oligo-T sequences. In addition, bPNA nucleases are cell membrane permeable and therefore may be used to target native intracellular substrates. In addition, these data indicate that bPNA scaffolds can be a platform for new synthetic binders to particular nucleic acid structural motifs.

Visualization of unstained homo/heterogeneous DNA nanostructures by low-voltage scanning transmission electron microscopy

Three-dimensional (3D) homo/heterogeneous DNA nanostructures were studied with low-voltage scanning transmission electron microscopy (LV-STEM). Four types of 3D DNA nanostructures were designed and fabricated by the origami method including newly proposed protocols. The low-energy electron probe and optimized dark-field STEM detector enabled individual unstained DNA nanostructures to be clearly imaged by the single acquisition without the averaging process. For the vertically stacked double structures, assembled through modified single-stranded domains, and the structures containing a square opening (i.e., a hole) in the center, the LV-STEM successfully reveals the vertical information of these 3D structures as the contrast differences compared to the reference. For the heterogeneous structures, the LV-STEM visualized both regions of the functionalized gold nanoparticles and the DNA base structure with distinct contrasts. This study introduces a straightforward method to fabricate stackable DNA nanostructures or nanoparticles by replacing a relatively small number of incumbent DNA strands, which could realize the simple and sophisticated fabrication of higher-order 3D DNA homo/hetero nanostructures. Together with these design techniques of DNA nanostructures, this study has demonstrated that the LV-STEM is the swift and simple method for visualizing the 3D DNA nanostructures and certifying the fabricated products as the specified design, which is applicable to various research fields on soft materials including DNA nanotechnology.

Nuclear body formation by Arabidopsis CPL1-RCF3 complex requires single-stranded RNA-binding domains

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) and REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) form a complex that function in RNA quality control under various abiotic and biotic stresses. Amino acid sequences show double-stranded RNA-binding motif 1(DRM1) and multiple K-homology (KH) domains in CPL1 and RCF3, respectively. Here we showed that both CPL1 and RCF3 specifically binds to single-stranded poly(C) RNA at DRM1 and multiple KH domains. We also showed that in planta, more than 3 KH domains are sufficient for RCF3 to form a complex with CPL1 in nuclear bodies. However, larger RCF3 fragments were necessary to form speckle structures, suggesting that polyvalent molecular interactions around RCF3 are essential for forming the nuclear structures necessary to process various precursor transcripts.

Design and Characterization of RNA Nanotubes.

RNA is a functionally rich and diverse biomaterial responsible for regulating several cellular processes. This functionality has been harnessed to build predominately small nanoscale structures for drug delivery and the treatment of disease. The understanding of design principles to build large RNA structures will allow for further control of stoichiometry and spatial arrangement drugs and ligands. We present the design and characterization of RNA nanotubes that self-assemble from programmable monomers, or tiles, formed by five distinct RNA strands. Tiles include double crossover junctions and assemble via single-stranded sticky-end domains. We find that nanotube formation is dependent on the intertile crossover distance. The average length observed for the annealed RNA nanotubes is ≈1.5 μm, with many nanotubes exceeding 10 μm, enabling the characterization of RNA nanotubes length distribution via fluorescence microscopy. Assembled tubes were observed to be stable for more than 24 h, however post-annealing growth under isothermal conditions does not occur. Nanotubes assemble also from RNA tiles modified to include a single-stranded overhang (toehold), suggesting that it may be possible to decorate these large RNA scaffolds with nanoparticles or other nucleic acid molecules.

Sequence-Dependent Effects of Monovalent Cations on the Structural Dynamics of Trinucleotide-Repeat DNA Hairpins.

Repetitive trinucleotide DNA sequences at specific genetic loci are associated with numerous hereditary, neurodegenerative diseases. The propensity of single-stranded domains containing these sequences to form secondary structure via extensive self-complementarity disrupts normal DNA processing to create genetic instabilities. To investigate these intrastrand structural dynamics, a DNA hairpin system was devised for single-molecule fluorescence study of the folding kinetics and energetics for secondary structure formation between two interacting, repetitive domains with specific numbers of the same trinucleotide motif (CXG), where X = T or A. Single-molecule fluorescence resonance energy transfer (smFRET) data show discrete conformational transitions between unstructured and closed hairpin states. The lifetimes of the closed hairpin states correlate with the number of repeats, with (CTG) N/(CTG) N domains maintaining longer-lived, closed states than equivalent-sized (CAG) N/(CAG) N domains. NaCl promotes similar degree of stabilization for the closed hairpin states of both repeat sequences. Temperature-based, smFRET experiments reveal that NaCl favors hairpin closing for (CAG) N/(CAG) N by preordering single-stranded repeat domains to accelerate the closing transition. In contrast, NaCl slows the opening transition of CTG hairpins; however, it promotes misfolded conformations that require unfolding. Energy diagrams illustrate the distinct folding pathways of (CTG) N and (CAG) N repeat domains and identify features that may contribute to their gene-destabilizing effects.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Differential Effects of Strand Asymmetry on the Energetics and Structural Flexibility of DNA Internal Loops.

Internal loops within structured nucleic acids disrupt local base stacking and destabilize neighboring helical domains; however, these structural motifs also expand the conformational and functional capabilities of structured nucleic acids. Variations in the size, distribution of loop nucleotides on opposing strands (strand asymmetry), and sequence alter their biophysical properties. Here, the thermodynamics and structural flexibility of oligo-T-rich DNA internal loops were systematically investigated in terms of loop size and strand asymmetry. From optical melting experiments, a thermodynamic prediction model is proposed for the energetic penalty of internal loops that accounts for diminishing enthalpic and increasing entropic contributions due to loop size and strand asymmetry for bulges, asymmetric loops, and symmetric loops. These single-stranded domains become less sequence-dependent and more polymeric as the loop size increases. Single-molecule fluorescence resonance energy transfer studies reveal a gradual transition in conformation and structural flexibility from an elongated domain to an increasingly flexible bend that results from increasing strand asymmetry. The findings provide a framework for understanding the thermodynamic and conformational effects of internal loops for the rational design of functional DNA nanostructures.

The sibling sRNAs NgncR_162 and NgncR_163 of Neisseria gonorrhoeae participate in the expression control of metabolic, transport and regulatory proteins.

Neisseria gonorrhoeae is the causative agent of gonorrhoea, the second most common bacterial sexually transmitted disease. Riboregulation mediated by small regulatory RNAs (sRNAs) is increasingly recognized as an important means of gene expression control in this human-restricted pathogen. sRNAs act at the post-transcriptional level by base-pairing with their target mRNAs which affects translation initiation and/or mRNA stability. In this study we initiated the characterization of a pair of highly conserved sRNAs of N. gonorrhoeae which exhibit redundant functions in the control of a common set of target genes. The identified targets of the sibling sRNAs NgncR_162 and NgncR_163 participate in basic metabolic processes including the methylcitrate and citrate cycle, aa uptake and degradation, and also in transcription regulation. Our data indicate that the sibling sRNAs control their targets via direct base-pairing between the same single-stranded domain(s) of the sRNA and the ribosome binding site in the 5'-untranslated region of the mRNA.

Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus.

This data article is related to the research article entitled “The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide” [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide.

The HIV-1 leader RNA is exquisitely sensitive to structural changes

The untranslated leader of the HIV-1 RNA genome is highly structured and contains multiple replication signals. We probed in detail the sequence requirements of a small single-stranded domain using a combination of in silico, in vitro and in vivo virus experiments. Although ‘structure-neutral’ mutations can be designed by RNA prediction algorithms, experimental follow-up studies nearly always demonstrate local or regional RNA structure changes. Our results indicate that the wild-type HIV-1 RNA sequence has been selected from total sequence space as a unique solution to present critical replication signals in the context of a complex leader structure with small intervening single-stranded segments.

Amplified and Multiplexed Detection of DNA Using the Dendritic Rolling Circle Amplified Synthesis of DNAzyme Reporter Units

The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg(2+)-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg(2+)-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg(2+)-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg(2+)-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals.

In Vivo SELEX of Single-Stranded Domains in the HIV-1 Leader RNA

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.

In vivo SELEX of single-stranded domains in the HIV-1 leader RNA

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.

Toehold-Mediated Nonenzymatic DNA Strand Displacement As a Platform for DNA Genotyping

Toehold-mediated DNA strand displacement provides unique advantages in the construction and manipulation of multidimensional DNA nanostructures as well as nucleic acid sequence analysis. We demonstrate a step change in the use of toehold-mediated DNA strand displacement reactions, where a double-stranded DNA duplex, containing a single-stranded toehold domain, enzymatically generated and then treated as a molecular target for analysis. The approach was successfully implemented for human DNA genotyping, such as gender identification where the amelogenin gene was used as a model target system, and detecting single nucleotide polymorphisms of human mitochondrial DNA. Kinetics of the strand displacement was monitored by the quenched Förster resonance energy transfer effect.

The A-nucleotide preference of HIV-1 in the context of its structured RNA genome

A bipartition of HIV-1 RNA genome sequences into single- and double-stranded nucleotides is possible based on the secondary structure model of a complete 9 kb genome. Subsequent analysis revealed that the well-known lentiviral property of A-accumulation is profoundly present in single-stranded domains, yet absent in double-stranded domains. Mutational rate analysis by means of an unrestricted model of nucleotide substitution suggests the presence of an evolutionary equilibrium to preserve this biased nucleotide distribution.

A structurally tunable DNA-based extracellular matrix.

The principles of DNA nanotechnology and protein engineering have been combined to generate a new class of artificial extracellular matrices. The potential of this material for ex vivo cellular scaffolding was demonstrated using experiments in which human cervical cancer cells were found to adhere strongly, stay alive, and grow with high migration rates. The use of DNA in our DNA/protein-based matrices makes these structures inherently amenable to structural tunability. By engineering single-stranded domains into the DNA portions, we were able to fine-tune the scaffold's persistence length and stiffness as perceived by cells. This was used to direct the outcome of the cell's cytoskeletal arrangement and overall shape, the status of its signal transduction protein p-FAK, and the localization of its intracellular transcription factors FOXO1a. This contribution lays the groundwork for the facile and modular construction of programmable extracellular matrices that can bring about the systematic study and replication of the naturally occurring extracellular niche.