DNA Hairpin Loops Research Articles

Hierarchical Mechanical Transduction of Precision-Engineered DNA Hydrogels with Sacrificial Bonds.

Engineering the response to external signals in mechanically switchable hydrogels is important to promote smart materials applications. However, comparably little attention has focused on embedded precision mechanisms for autonomous nonlinear response in mechanical profiles in hydrogels, and we lack understanding of how the behavior from the molecular scale transduces to the macroscale. Here, we design a nonlinear stress-strain response into hydrogels by engineering sacrificial DNA hairpin loops into model network hydrogels formed from star-shaped building blocks. We characterize the force-extension response of single DNA hairpins and are able to describe how the specific topology influences the nonlinear mechanical behavior at different length scales. For this purpose, we utilize force spectroscopy as well as microscopic and macroscopic deformation tests. This study contributes to a better understanding of designing nonlinear strain-adaptive features into hydrogel materials.

Target-Catalyzed Self-Assembly of DNA-Streptavidin Nanogel for Enzyme-Free miRNA Assay.

Rapid, sensitive, specific, and user-friendly microRNA (miRNA) assays are in high demand for point-of-care diagnosis. Target-catalyzed toehold-mediated strand displacement (TMSD) has received increasing attention as an enzyme-free molecular tool for DNA detection. However, the application of TMSD to miRNA targets is challenging because relatively weak DNA/RNA hybridization leads to failure in the subtle kinetic control of multiple hybridization steps. Here, a simple method is presented for miRNA assay based on the one-pot self-assembly of Y-shaped DNAs with streptavidin via an miRNA-catalyzed TMSD cascade reaction. A single miRNA catalyzes the opening cycle of DNA hairpin loops to generate multiple Y-shaped DNAs carrying biotin and a quencher at the end of their arms. Introducing a single base-pair mismatch near the toehold facilitates RNA-triggered strand displacement while barely disturbing nonspecific reactions. The Y-shaped DNAs are self-assembled with fluorescently labeled streptavidin (sAv), which produces nanoscale DNA-sAv nanogel particles mediating efficient Förster resonance energy transfer in their 3D network. The enhancing effect dramatically reduces the detection limit from the nanomolar level to the picomolar level. This work proves that TMSD-based DNA nanogel with a base-pair mismatch incorporated to a hairpin structure is a promising approach towards sensitive and accurate miRNA assay.

Modulating the Fluorescence of Silver Nanoclusters Wrapped in DNA Hairpin Loops via Confined Strand Displacement and Transient Concatenate Ligation for Amplifiable Biosensing.

It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.

Short DNA-catalyzed formation of quantum dot-DNA hydrogel for enzyme-free femtomolar specific DNA assay

Fast, sensitive, specific, and user-friendly DNA assay is a key technique for the next generation point-of-care molecular diagnosis. However, high-cost, time-consuming, and complicated enzyme-based DNA amplification step is essential to achieve high sensitivity. Herein, a short target DNA-catalyzed formation of quantum dot (QD)-DNA hydrogel is proposed as a new DNA assay platform satisfying the above requirements. A single-stranded target DNA catalyzes the opening cycle of DNA hairpin loops, which are quickly self-assembled with DNA-functionalized QDs to generate QD-DNA hydrogel. The three-dimensional hydrogel network allows efficient resonance energy transfer, dramatically lowering the limit of detection down to ~6 fM without enzymatic DNA amplification. The QD-DNA hydrogel also enables a rapid detection (1 h) with high specificity even for a single-base mismatch. The clinical applicability of the QD-DNA hydrogel is demonstrated for the Klebsiella pneumoniae carbapenemase gene, one of the key targets of drug-resistant pathogenic bacteria.

An extended APOBEC3A mutation signature in cancer

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

The Application of Tetrahedral Framework Nucleic Acids as a Drug Carrier in Biomedicine Fields.

In recent years, tetrahedral Framework Nucleic Acids(tFNAs) have become a hot topic in the field of DNA nanostructures because of their stable structures, nanoscale size, superior mechanical properties and convenient synthesis with high yield. tFNAs are considered promising drug delivery carriers because they can pass through the cellular membrane without any help and they have a good biocompatibility and biodegradability. Besides, they have rich modification sites, they can be modified by kinds of functional groups. The functionalization molecules can be modified on the vertexes, embedded between the double-stranded DNA of the tetrahedron edges, hanged on the edges, or encapsulated in the cage-like structure of the tetrahedron. The structure of tetrahedron can also be intelligently controlled through smart design, such as integrating DNA hairpin loop structure onto the edges. Nowadays, DNA tetrahedron will have a broader development prospect in the application of drug transport carriers and intelligent drug carriers. Therefore, DNA material is a new carrier material with great advantages and has a very broad application prospect in the construction of an intelligent drug transport system.

Stabilization of DNA Loop Structures by Large Cations.

The DNA-binding properties of large cations differ from those of metal ions due to steric exclusion from base-paired regions. In this study, the thermal stability of DNA secondary structures, including duplexes, internal loops, bulge loops, hairpin loops, dangling ends, and G-quadruplexes, was investigated in the presence of cations of different sizes. Large cations, such as tetrabutylammonium and tetrapentylammonium ions, reduced the stability of fully matched duplexes but increased the stability of duplexes with a long loop. The cations also increased the stability of G-quadruplexes with a long loop, and the degree of stabilization was greater for low-stability G-quadruplexes. Analysis of the salt concentration dependence indicates that large cations bind to the loop nucleotides, leading to counteracting the destabilization effect on base pairing. It is likely that binding occurs when loop nucleotides are sufficiently flexible to allow for greater accessibility for large cations. These results provide insight into nucleic acid interactions with large cationic molecules and suggest a potential method for stabilizing noncanonical DNA structures under intracellular conditions.

Quantitative Binding of Divalent Metal Ions to DNA Hairpin Loops

Quantitative Binding of Divalent Metal Ions to DNA Hairpin Loops

Structural and Mechanistic Implications of Rearrangement Frequencies within Human TCRBV Genes.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

The Mismatch-Binding Factor MutSβ Can Mediate ATR Activation in Response to DNA Double-Strand Breaks

Ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase, a master regulator of DNA-damage response, is activated by RPA-coated single-stranded DNA (ssDNA) generated at stalled replication forks or DNA double-strand breaks (DSBs). Here, we identify the mismatch-binding protein MutSβ, a heterodimer of MSH2 and MSH3, as a key player in this process. MSH2 and MSH3 form a complex with ATR and its regulatory partner ATRIP, and their depletion compromises the formation of ATRIP foci and phosphorylation of ATR substrates in cells responding to replication-associated DSBs. Purified MutSβ binds to hairpin loop structures that persist in RPA-ssDNA complexes and promotes ATRIP recruitment. Mutations in the mismatch-binding domain of MSH3 abolish the binding of MutSβ to DNA hairpin loops and its ability to promote ATR activation by ssDNA. These results suggest that hairpin loops might form in ssDNA generated at sites of DNA damage and trigger ATR activation in a process mediated by MutSβ.

Targeting and function of proteins mediating translation initiation in organelles of Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has two translationally active organelles - the apicoplast and mitochondrion, which import nuclear-encoded translation factors to mediate protein synthesis. Initiation of translation is a complex step wherein initiation factors (IFs) act in a regulated manner to form an initiation complex. We identified putative organellar IFs and investigated the targeting, structure and function of IF1, IF2 and IF3 homologues encoded by the parasite nuclear genome. A single PfIF1 is targeted to the apicoplast. Apart from its critical ribosomal interactions, PfIF1 also exhibited nucleic-acid binding and melting activities and mediated transcription anti-termination. This suggests a prominent ancillary function for PfIF1 in destabilisation of DNA and RNA hairpin loops encountered during transcription and translation of the A+T rich apicoplast genome. Of the three putative IF2 homologues, only one (PfIF2a) was an organellar protein with mitochondrial localisation. We additionally identified an IF3 (PfIF3a) that localised exclusively to the mitochondrion and another protein, PfIF3b, that was apicoplast targeted. PfIF3a exhibited ribosome anti-association activity, and monosome splitting by PfIF3a was enhanced by ribosome recycling factor (PfRRF2) and PfEF-G(Mit). These results fill a gap in our understanding of organellar translation in Plasmodium, which is the site of action of several anti-malarial compounds.

Kinetics of conformational fluctuations in DNA hairpin-loops in crowded fluids

Biochemical reactions in crowded fluids have been predicted to differ strongly from those in aqueous solutions. Both excluded-volume interactions with surrounding macromolecules and crowding-induced subdiffusion have been predicted to stabilize complex formation of reaction partners. Here we show that both facets of crowding strongly alter the stochastic opening and closing of a single-stranded DNA hairpin. Crowding not only slows down the kinetics but also increases the steady-state fraction of closed hairpins significantly. The latter is shown to be enhanced if crowding is associated with subdiffusion. Biochemical reactions in crowded fluids are therefore sensistive to both excluded-volume interactions and changes of the reactants' diffusion characteristics.

Single-stranded DNA loops as fiducial markers for exploring DNA–protein interactions in single molecule imaging

A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end of linear double-stranded (ds) DNA templates was developed. The loop structure serves as a fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied to study DNA–protein interactions. The nucleic acid end-labels allow discrimination of the polarity of the DNA template in the AFM while limiting non-specific interactions which might occur from non-nucleic acid labels. Homo-polynucleotide ssDNA loops made up of 20 base-pairs (bp) for each of the four bases (A, T, G, C) were investigated to determine the effects of sequence on template labelling. The products were produced with high efficiency and high yield with the loop readily distinguished from the dsDNA template by height and diameter in the AFM. The application of the method to study DNA transcription was investigated by firing Escherichia Coli RNA polymerase (RNAP) from a λPR promoter in the direction of the loop-labelled end. The ssDNA loops captured elongating complexes of RNAP, arresting transcription and preventing dissociation. The dual role of the loop as a polarity marker and retainer of previously active RNAP will allow mechanisms of gene expression to be studied with single molecule sensitivity by AFM. This will enable insight into molecular interactions of RNAP on single DNA templates in convergent or tandem transcription configurations.

Label-Free and Substrate-Free Potentiometric Aptasensing Using Polycation-Sensitive Membrane Electrodes

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

A DNA based five-state switch with programmed reversibility

A programmable switch based on a DNA hairpin loop is functionalised with a rigid or flexible porphyrin or FAM and TAMRA FRET pair, which provides insight into the restructuring of the hairpin as well as porphyrin-porphyrin coupling. The switch contains five discrete states which can be accessed independently and followed by real-time spectroscopy, opening the way to a quinary computing code.

Highly efficient remote controlled release system based on light-driven DNA nanomachine functionalized mesoporous silica

An intelligent photoswitchable single-molecule nanomachine with DNA hairpin-loop structure was designed by the incorporation of azobenzene groups in DNA sequences, which was studied by fluorescence resonance energy transfer (FRET) and attached onto the surface of mesoporous silica. Based on the photo-induced conformational transformation of DNA, highly efficient controlled release was realized.

Effect of DNA hairpin loops on the twist of planar DNA origami tiles.

The development of scaffolded DNA origami, a technique in which a long single-stranded viral genome is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides, represents an important milestone in DNA nanotechnology. Recent findings have revealed that two-dimensional (2D) DNA origami structures based on the original design parameters adopt a global twist with respect to the tile plane, which may be because the conformation of the constituent DNA (10.67 bp/turn) deviates from the natural B-type helical twist (10.4 bp/turn). Here we aim to characterize the effects of DNA hairpin loops on the overall curvature of the tile and explore their ability to control, and ultimately eliminate any unwanted curvature. A series of dumbbell-shaped DNA loops were selectively displayed on the surface of DNA origami tiles with the expectation that repulsive interactions among the neighboring dumbbell loops and between the loops and the DNA origami tile would influence the structural features of the underlying tiles. A systematic, atomic force microscopy (AFM) study of how the number and position of the DNA loops influenced the global twist of the structure was performed, and several structural models to explain the results were proposed. The observations unambiguously revealed that the first generation of rectangular shaped origami tiles adopt a conformation in which the upper right (corner 2) and bottom left (corner 4) corners bend upward out of the plane, causing linear superstructures attached by these corners to form twisted ribbons. Our experimental observations are consistent with the twist model predicted by the DNA mechanical property simulation software CanDo. Through the systematic design and organization of various numbers of dumbbell loops on both surfaces of the tile, a nearly planar rectangular origami tile was achieved.

Ligand‐Assisted Complex Formation of Two DNA Hairpin Loops

All loopy: The interaction of a series of mismatch-binding molecules with hairpin DNA that contains a d(CGG)3 sequence in the loop is described (see picture). Native polyacrylamide gel electrophoresis of hairpin-loop DNA shows that the newly synthesized mismatch-binding molecule, a tetrameric form of N-methoxycarbonyl-1,8-naphthyridine, assists the formation of a loop–loop complex of two DNA hairpin loops.

Ligand‐Assisted Complex Formation of Two DNA Hairpin Loops

Voller Schleifen: Die Wechselwirkung einer Reihe von fehlpaarenden Molekülen mit Haarnadelschleifen-DNA, die eine d(CGG)3-Sequenz in der Schleife enthält, wird beschrieben (siehe Bild). Native Polyacrylamid-Gelelektrophorese von Haarnadelschleifen-DNA zeigt, dass das neu synthetisierte fehlpaarende Molekül, eine tetramere Form von N-Methoxycarbonyl-1,8-naphthyridin, an der Bildung eines Schleifen-Schleifen-Komplexes zwischen zwei DNA-Haarnadelschleifen beteiligt ist. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Luminescence Resonance Energy Transfer in Heterodinuclear LnIII Complexes for Sensing Biologically Relevant Anions

AbstractDinuclear lanthanide(III) complexes of the macrocycles 1,4‐bis[1‐4,7,10‐ tris(carbamoylmethyl)‐1,4,7,10‐tetraazacyclododecane]‐m‐xylene (1) and 1,3‐bis[1‐4,7,10‐tris(carbamoylmethyl)‐1,4,7,10‐tetraazacyclododecane]‐p‐xylene (2) with LnIII = EuIII, TbIII were prepared. Studies using direct excitation (7F0 → 5D0) europium(III) luminescence spectroscopy show that several anions including phosphate, methylphosphate, double‐stranded DNA, a DNA hairpin loop, and fluoride bind to both EuIII centers in Eu2(1) in solutions at pH 7.0, 0.100 M NaNO3. Recovered luminescence lifetime data is consistent with replacement of water ligands by these anions. There are distinct changes in the relative intensities of EuIII emission peaks for complexes of Eu2(1) and Eu2(2) with phosphate, fluoride, carbonate, and phosphate ester ligands. This suggests that the EuIII dinuclear complexes show promise as ratiometric sensors for these anions. In contrast, the emission peak ratios for the Tb2(1) and Tb2(2) complex show a lower response upon binding of each anionic ligand. Luminescence resonance energy transfer (LRET) studies with mixed EuIII/NdIII complexes show that the phosphate complexes of Ln2(1) or Ln2(2) both have LnIII–LnIII internuclear distances of 7.7 ± 0.9 Å and the methylphosphate complexes of Ln2(1) and Ln2(2) have internuclear distances of 7.8 ± 0.6 and 8.0 ± 0.7 Å, respectively. LRET experiments also show the two lanthanide ion centers in Ln2(2) are 8.4 ± 0.5 Å apart when bound to GC rich duplex DNA. None of the other anions (carbonate, fluoride, DNA hairpins) form complexes that show measurable energy transfer between the lanthanide ions, which is consistent with intranuclear distances >8 Å.