Single-stranded DNA Oligomers Research Articles

Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities.

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells.

Aptamer-Based Probes for Cancer Diagnostics and Treatment.

Aptamers are single-stranded DNA or RNA oligomers that have the ability to generate unique and diverse tertiary structures that bind to cognate molecules with high specificity. In recent years, aptamer researches have witnessed a huge surge, owing to its unique properties, such as high specificity and binding affinity, low immunogenicity and toxicity, and simplicity of synthesis with negligible batch-to-batch variation. Aptamers may bind to targets, such as various cancer biomarkers, making them applicable for a wide range of cancer diagnosis and treatment. In cancer diagnostic applications, aptamers are used as molecular probes instead of antibodies. They have the potential to detect various cancer-associated biomarkers. For cancer therapeutic purposes, aptamers can serve as therapeutic or delivery agents. The chemical stabilization and modification strategies for aptamers may expand their serum half-life and shelf life. However, aptamer-based probes for cancer diagnosis and therapy still face several challenges for successful clinical translation. A deeper understanding of nucleic acid chemistry, tissue distribution, and pharmacokinetics is required in the development of aptamer-based probes. This review summarizes their application in cancer diagnostics and treatments based on different localization of target biomarkers, as well as current challenges and future prospects.

Biochemical analysis of the replication initiator protein of staphylococcal plasmid pC194

The staphylococcal plasmid pC194 is replicated through the rolling-circle mechanism. Its replication protein RepA has been proposed to initiate replication by making a bond between Y214 and DNA phosphate via transesterification and to terminate it by hydrolyzing DNA with E210 and carrying out strand transfer. We tested this model by examining the catalytic functions of the protein with purified RepA proteins and single-stranded DNA oligomers. The wild-type RepA formed a covalent bond with the DNA phosphate at the predicted initiation site. It hydrolyzed the phosphodiester bond at the site, which activity was found to depend on the presence of a large pseudopalindrome contained in the replication origin. The protein carried out a strand-transfer reaction which mimicked the termination step of replication. A Y214F and an E210A mutant respectively lacked the transesterification and the hydrolytic activity. These results are consistent with the previously proposed model, which was based solely on molecular genetics results. In addition, an E142A mutant was found to lack both activities, suggesting that the residue may coordinate the divalent cation necessary for them. A possible role of the pseudopalindrome in controlling the two activities of RepA during a replication cycle is also discussed.

Heme binding to cold shock protein D, CspD, from Vibrio cholerae

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.

Quantifying inter-organelle membrane contact sites using proximity ligation assay in fixed optic nerve sections

Membrane contact sites (MCS) play crucial roles in cell physiology with dysfunction in several MCS proteins being linked with neurological and optic nerve diseases. Although there have been significant advances in imaging these interactions over the past two decades with advanced electron microscopy techniques, super-resolution imaging and proximity-dependent fluorescent reporters, a technique to observe and quantify MCS in mammalian optic nerve tissues has not yet been reported. We demonstrate for the first time that proximity ligation assay (PLA), a technique already used in mammalian cell lines, can be used as an efficient method of quantifying inter-organelle contact sites, namely mitochondria-endoplasmic reticulum (ER) and mitochondria-late-endosomes, in mammalian optic nerve tissues treated with adeno-associated virus (AAV) gene therapy with wild-type or phosphomimetic (active) protrudin. PLA utilises complementary single-stranded DNA oligomers bound to secondary antibodies that hybridise and complete a circular piece of DNA when the primary antibodies of interest interact. These interactions can be detected by amplifying the circular DNA and adding fluorescent probes. We show that PLA is a useful method that can be used to quantify MCS in optic nerve tissues. We have found that upregulation of protrudin with gene therapy significantly increases the number of mitochondria-ER and mitochondria-Rab7-late endosomes contact sites in optic nerves.

Dye Quenching of Carbon Nanotube Fluorescence Reveals Structure-Selective Coating Coverage.

Many properties and applications of single-wall carbon nanotubes (SWCNTs) depend strongly on the coatings that allow their suspension in aqueous media. We report that SWCNT fluorescence is quenched by reversible physisorption of dye molecules such as methylene blue, and that measurements of that quenching can be used to infer structure-specific exposures of the nanotube surface to the surrounding solution. SWCNTs suspended in single-stranded DNA oligomers show quenching dependent on the combination of nanotube structure and ssDNA base sequence. Several sequences are found to give notably high or low surface coverages for specific SWCNT species. These effects seem correlated with the selective recognitions used for DNA-based structural sorting of nanotubes. One notable example is that dye quenching of fluorescence from SWCNTs coated with the (ATT)4 base sequence is far stronger for one (7,5) enantiomer than for the other, showing that coating coverage is associated with the coating affinity difference reported previously for this system. Equilibrium modeling of quenching data has been used to extract parameters for comparative complexation constants and accessible surface areas. Further insights are obtained from molecular dynamics simulations, which give estimated contact areas between ssDNA and SWCNTs that correlate with experimentally inferred surface exposures and account for the enantiomeric discrimination of (ATT)4.

Structural and Photophysical Templating of Conjugated Polyelectrolytes with Single-Stranded DNA.

A promising approach to influence and control the photophysical properties of conjugated polymers is directing their molecular conformation by templating. We explore here the templating effect of single-stranded DNA oligomers (ssDNAs) on cationic polythiophenes with the goal to uncover the intermolecular interactions that direct the polymer backbone conformation. We have comprehensively characterized the optical behavior and structure of the polythiophenes in conformationally distinct complexes depending on the sequence of nucleic bases and addressed the effect on the ultrafast excited-state relaxation. This, in combination with molecular dynamics simulations, allowed us a detailed atomistic-level understanding of the structure–property correlations. We find that electrostatic and other noncovalent interactions direct the assembly with the polymer, and we identify that optimal templating is achieved with (ideally 10–20) consecutive cytosine bases through numerous π-stacking interactions with the thiophene rings and side groups of the polymer, leading to a rigid assembly with ssDNA, with highly ordered chains and unique optical signatures. Our insights are an important step forward in an effective approach to structural templating and optoelectronic control of conjugated polymers and organic materials in general.

Conformations and cryo-force spectroscopy of spray-deposited single-strand DNA on gold

Cryo-electron microscopy can determine the structure of biological matter in vitrified liquids. However, structure alone is insufficient to understand the function of native and engineered biomolecules. So far, their mechanical properties have mainly been probed at room temperature using tens of pico-newton forces with a resolution limited by thermal fluctuations. Here we combine force spectroscopy and computer simulations in cryogenic conditions to quantify adhesion and intra-molecular properties of spray-deposited single-strand DNA oligomers on Au(111). Sub-nanometer resolution images reveal folding conformations confirmed by simulations. Lifting shows a decay of the measured stiffness with sharp dips every 0.2–0.3 nm associated with the sequential peeling and detachment of single nucleotides. A stiffness of 30–35 N m−1 per stretched repeat unit is deduced in the nano-newton range. This combined study suggests how to better control cryo-force spectroscopy of adsorbed heterogeneous (bio)polymer and to potentially enable single-base recognition in DNA strands only few nanometers long.

Deposition of sticky spheres in channel flow: Modeling of surface coverage evolution requires accurate sphere-sphere collision hydrodynamics

We analyzed the role of hydrodynamic interactions in a microfluidic channel flow containing a dilute suspension of micron-scale colloidal spheres (0.03%, 0.1%, 0.3% volume fraction) engineered to adhere onto a collector patch on the channel wall at wall shear rates of 9.3–930 s−1. Particle-wall adhesion was mediated by single-stranded DNA oligomers grafted onto the spheres and the glass channel wall, producing well-defined interactions via DNA strand base pairing. Particle positions in the flow were evolved using Brownian dynamics simulations in which hydrodynamic interactions between moving particles and the channel walls and/or adhered particles were computed off-line using a series of local simulations that explicitly resolve the fluid flow at the particle scale. By systematically varying the nature of hydrodynamic interactions captured in the Brownian dynamics simulations, we find that the interactions between moving and adhered particles represents the single most important physical element in such models. Once captured sufficiently accurately, the resulting models are able to predict coarse variables such as the overall particle coverage evolution, as well as more subtle characteristics, such as the microstructural distribution of the adhered particles.

Adsorption Mechanisms of Nucleobases on the Hydrated Au(111) Surface.

The solution environment is of fundamental importance in the adsorption of molecules on surfaces, a process that is strongly affected by the capability of the adsorbate to disrupt the hydration layer above the surface. Here we disclose how the presence of interface water influences the adsorption mechanism of DNA nucleobases on a gold surface. By means of metadynamics simulations, we describe the distinctive features of a complex free-energy landscape for each base, which manifests activation barriers for the adsorption process. We characterize the different pathways that allow each nucleobase to overcome the barriers and be adsorbed on the surface, discussing how they influence the kinetics of adsorption of single-stranded DNA oligomers with homogeneous sequences. Our findings offer a rationale as to why experimental data on the adsorption of single-stranded homo-oligonucleotides do not straightforwardly follow the thermodynamics affinity rank.

Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

Functionalisation of Detonation Nanodiamond for Monodispersed, Soluble DNA-Nanodiamond Conjugates Using Mixed Silane Bead-Assisted Sonication Disintegration

Nanodiamonds have many attractive properties that make them suitable for a range of biological applications, but their practical use has been limited because nanodiamond conjugates tend to aggregate in solution during or after functionalisation. Here we demonstrate the production of DNA-detonation nanodiamond (DNA-DND) conjugates with high dispersion and solubility using an ultrasonic, mixed-silanization chemistry protocol based on the in situ Bead-Assisted Sonication Disintegration (BASD) silanization method. We use two silanes to achieve these properties: (1) 3-(trihydroxysilyl)propyl methylphosphonate (THPMP); a negatively charged silane that imparts high zeta potential and solubility in solution; and (2) (3-aminopropyl)triethoxysilane (APTES); a commonly used functional silane that contributes an amino group for subsequent bioconjugation. We target these amino groups for covalent conjugation to thiolated, single-stranded DNA oligomers using the heterobifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The resulting DNA-DND conjugates are the smallest reported to date, as determined by Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). The functionalisation method we describe is versatile and can be used to produce a wide variety of soluble DND-biomolecule conjugates.

Single-stranded DNA oligomer brush structure is dominated by intramolecular interactions mediated by the ion environment.

Single-stranded DNA (ssDNA) brushes, in which ssDNA oligomers are tethered to surfaces in dense monolayers, are being investigated for potential biosensing applications. The structure of the brush can affect the selectivity and the hybridization efficiency of the device. The structure is commonly thought to result from the balance of intramolecular interactions, intermolecular interactions within the monolayer, and molecule-surface interactions. Here, we test the hypothesis that ssDNA oligomer brush structure is dominated by intramolecular interactions. We use AFM to measure the height of an ssDNA brush and molecular dynamics to simulate the end-to-end distance, both as a function of ionic strength of the surrounding solution. The brush height and the molecule end-to-end distance match quantitatively, providing evidence that the brush structure is dominated by intramolecular interactions (mediated by ions). The physical basis of the intramolecular interactions is elucidated by the simulations.

Interaction Heterogeneity can Favorably Impact Colloidal Crystal Nucleation.

Colloidal particles with short-ranged attractions, e.g., micron-scale spheres functionalized with single-stranded DNA oligomers, are susceptible to becoming trapped in disordered configurations even when a crystalline arrangement is the ground state. Moreover, for reasons that are not well understood, seemingly minor variations in the particle formulation can lead to dramatic changes in the crystallization outcome. We demonstrate, using a combination of equilibrium and nonequilibrium computer simulations, that interaction heterogeneity-variations in the energetic interactions among different particle pairs in the population-may favorably impact crystal nucleation. Specifically, interaction heterogeneity is found to lower the free energy barrier to nucleation via the formation of clusters comprised preferentially of strong-binding particle pairs. Moreover, gelation is inhibited by "spreading out over time" the nucleation process, resulting in a reduced density of stable nuclei, allowing each to grow unhindered and larger. Our results suggest a simple and robust approach for enhancing colloidal crystallization near the "sticky sphere" limit, and support the notion that differing extents of interaction heterogeneity arising from various particle functionalization protocols may contribute to the otherwise unexplained variations in crystallization outcomes reported in the literature.

Selective Binding of Thioflavin T in Sequence‐Exchanged Single Strand DNA Oligomers and Further Interaction with Phospholipid Membranes

AbstractSelectivity of ThT towards sequence exchanged single strand DNA (ssDNA) oligomers in absence and presence of surface charged lipid vesicles has been studied in detail. The dye shows special affinity for TCTTA sequence with its –N(CH3)3 structural counterpart and π‐interaction with adenine in 5′‐CCCTCTTAACCC‐3′ (TC4). However, fluorescence spectroscopy and molecular docking suggest that the dye is perfectly aligned with the TAA sequence in 5′‐CCCTTAATCCCC‐3′ (TT4). Interactions with cationic and anionic lipid vesicles were studied to understand the biological relevance in specificity of ThT binding to ssDNA oligomers. Fluorescence intensity of ThT increases significantly due to interaction with charged liposomes along with a considerable hypsochromic shift. Interestingly, evolution of a shoulder at 465 nm was observed solely in cationic lipid membranes due to ThT‐dimer formation. It is concluded that the ssDNA bound ThT penetrates the hydrophobic bilayer of the anionic vesicles. However, they attach superficially to the cationic vesicles facilitating dimer formation. Exchange of the nucleotide sequence in ssDNA led to change in the excited state lifetime of bound ThT in anionic liposomes.

The colorimetric determination of selectively cleaved adenosines and guanosines in DNA oligomers using bicinchoninic acid and copper.

Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this proof-of-principle study, we demonstrate that bicinchoninic acid (BCA) and copper, when combined with purine-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of adenosines and/or guanosines in single-stranded DNA oligomers, even in the presence of pyrimidines. Furthermore, the reactions are stoichiometric, which allows for the quantification of the number of adenosines and/or guanosines in these oligomers. Because the BCA/copper reagent detects the reducing sugar, 2-deoxyribose, that results from the chemical cleavage of a given nucleotide's N-glycosidic bond, these colorimetric assays are effectively detecting apurinic sites in DNA oligomers, which are known to occur via DNA damage in biological systems. We demonstrate that simple digital analysis of the color-changing chromophore (BCA/copper) is all that is necessary to obtain quantifiable and reproducible data, which indicates that these assays should be broadly accessible.

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein.

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4-DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3-DNA complex.

Interaction of Polymers with Single-Wall Carbon Nanotubes

Polymers are widely used for postsynthesis processing, purification, and individualization of single-wall carbon nanotubes (SWNTs) in aqueous or organic solvent environments. Here, the interaction of single-stranded DNA oligomers (ssDNA) and of a polyfluorene copolymer (F8T2) with (6,5) SWNTs was investigated from desorption kinetics and in the case of ssDNA also using adsorption isotherms. Eyring analysis of desorption rate constants reveals a linear increase of activation enthalpies with ssDNA oligomer length until ΔdesH‡ saturates at (155 ± 5) kJ·mol–1 for oligomers exceeding the ssDNA Kuhn length of about 6 nm. The Gibbs energy for desorption of ΔdesG‡ = (96 ± 1) kJ·mol–1 is length-independent because of entropy–enthalpy compensation. The saturation of desorption energies at the high polymer coverages studied here is attributed to incomplete adsorption with typically no more than a single Kuhn segment of a polymer attached to a SWNT.

Highly Sensitive and Selective Surface-Enhanced Raman Spectroscopy Label-free Detection of 3,3',4,4'-Tetrachlorobiphenyl Using DNA Aptamer-Modified Ag-Nanorod Arrays.

An improved label-free approach for highly sensitive and selective detection of 3,3',4,4'-tetrachlorobiphenyl (PCB-77), a type of polychlorinated biphenyl, via surface-enhanced Raman spectroscopy (SERS) using DNA aptamer-modified Ag-nanorod arrays as the effective substrate is reported. The devised system consists of Ag-nanorod (Ag-NR) arrays with the PCB-77 binding aptamers anchored covalently to the Ag surfaces through a thiol linker. The aptamers are made of single-stranded DNA (ssDNA) oligomers, with one end standing on the Ag surface, and upon conjugation with PCB-77, the ssDNA molecules can change their conformation to hairpin loops, so that the Raman intensity of guanines at the other end of the DNA strand increases accordingly. As such, the intensity ratio I(656 cm(-1))/I(733 cm(-1)) increases concomitantly with the increase of the concentration of PCB-77, making the quantitative evaluation of trace amounts of PCB-77 attainable. Moreover, it is found that the DNA aptamer-based Ag-NR arrays can be more responsive with a lower and optimal density of the DNA molecules modified on the substrate surface, and the best sensitivity for detection of PCB-77 can be achieved with the lower detection limit approaching 3.3 × 10(-8) M. This work therefore demonstrates that the design of aptamer-modified Ag-NRs can be used as a practically promising SERS substrate for label-free trace detection of persistent organic pollutants (POPs) in the environment.

Exploring ion induced folding of a single-stranded DNA oligomer from molecular simulation studies.

One crucial issue in DNA hydration is the effect of salts on its conformational features. This has relevance in biology as cations present in the cellular environment shield the negative charges on the DNA backbone, thereby reducing the repulsive force between them. By screening the negative charges along the backbone, cations stabilize the folded structure of DNA. To study the effect of the added salt on single-stranded DNA (ss-DNA) conformations, we have performed room temperature molecular dynamics simulations of an aqueous solution containing the ss-DNA dodecamer with the 5'-CGCGAATTCGCG-3' sequence in the presence of 0.2, 0.5, and 0.8 M NaCl. Our calculations reveal that in the presence of the salt, the DNA molecule forms more collapsed coil-like conformations due to the screening of negative charges along the backbone. Additionally, we demonstrated that the formation of an octahedral inner-sphere complex by the strongly bound ion plays an important role in the stabilization of such folded conformation of DNA. Importantly, it is found that ion-DNA interactions can also explain the formation of non-sequential base stackings with longer lifetimes. Such non-sequential base stackings further stabilize the collapsed coil-like folded form of the DNA oligomer.