Unmodified Single-stranded DNA Research Articles

Development of a New Route for the Immobilization of Unmodified Single-Stranded DNA on Chitosan Beads and Detection of Released Guanine after Hydrolysis

In this work, chitosan beads were used as a cost-effective platform for the covalent immobilization of unmodified single-stranded DNA, using glutaraldehyde as a cross-linking agent. The immobilized DNA capture probe was hybridized in the presence of miRNA-222 as a complementary sequence. The target was evaluated based on the electrochemical response of the released guanine, using hydrochloride acid as a hydrolysis agent. Differential pulse voltammetry technique and screen-printed electrodes modified with COOH-functionalized carbon black were used to monitor the released guanine response before and after hybridization. The functionalized carbon black provided an important signal amplification of guanine compared to the other studied nanomaterials. Under optimal conditions (6 M HCl at 65 °C for 90 min), an electrochemical-based label-free genosensor assay exhibited a linear range between 1 nM and 1 µM of miRNA-222, with a detection limit of 0.2 nM of miRNA-222. The developed sensor was successfully used to quantify miRNA-222 in a human serum sample.

A pH-responsive bioassay for sensitive colorimetric detection of adenosine triphosphate based on switchable DNA aptamer and metal ion–urease interactions

A facile and economic colorimetric strategy was designed for ATP detection by rationally using urease, a pH-responsive molecule, and a metal-mediated switchable DNA probe. By utilizing metal ions as a modulator of urease activity, the concentration of ATP is translated into pH change, which can be readily visualized by naked eye. An unmodified single-stranded DNA probe was designed, which consists of a target binding sequence and two flanked cytosine (C)-rich sequences. This C-rich single-stranded DNA can form a hairpin structure triggered by Ag+ ions via C-Ag+-C base mismatch. Upon introduction of ATP, Ag+-coordinated hairpin DNA structure will be broken and release the included Ag+, thus inhibiting the activity of urease. Conversely, urease can hydrolyze urea and raise pH value of the solution, resulting in the color change of the sensing solution. The proposed assay allows determination of ATP as low as 1.6nM and shows a satisfactory result in human serum. Because of simple operation and low cost of this method, we believe it has a potential in point-of-care (POC) testing in resource-limited areas. Schematic illustration of pH-responsive colorimetric sensor for ATP detection based on switchable DNA aptamer and metal ion-urease interactions.

Sequence-Directed Covalent Protein-DNA Linkages in a Single Step Using HUH-Tags.

We present a robust strategy to covalently link proteins and DNA using HUH-endonuclease domains as fusion partners (HUH-tags). We show that HUH-tags react robustly with specific sequences of unmodified single-stranded DNA, and we have identified five tags that react orthogonally with distinct DNA sequences. We demonstrate the versatility of HUH-tags as fusion partners in Cas9-mediated gene editing and the construction of doubly DNA-tethered proteins for single-molecule studies. Finally we demonstrate application to cellular imaging in live and fixed cells.

Sequence Directed Formation of Covalent Protein-DNA Bonds

We have developed a novel method for linking proteins to DNA based on the HUH endonuclease family. HUH endonucleases are involved in replication of ssDNA in viruses and mobile bacterial plasmids. The HUH domain of the endonuclease nicks ssDNA at a specific sequence near the origin of replication, resulting in formation of a covalent phosphotyrosine intermediate between the DNA and catalytic tyrosine in the HUH protein. We have adapted these nucleases into small, soluble protein tags which robustly react with unmodified single-stranded DNA to form stable covalent bonds. We have shown that five of these proteins react with fully orthogonal DNA sequences and can be used for “one-pot”, site-specific labeling of complex structures. Three tags have shown robust activity in cultured mammalian cells, allowing single-step DNA-based fluorescent labeling of fusion proteins. We believe that this technology can expand the possibilities for DNA nanotechnology, both in vitro and in cultured cells.

Phosphorothioate DNA Stabilized Fluorescent Gold and Silver Nanoclusters.

Unmodified single-stranded DNA has recently gained popularity for the templated synthesis of fluorescent noble metal nanoclusters (NCs). Bright, stable, and biocompatible clusters have been developed primarily through optimization of DNA sequence. However, DNA backbone modifications have not yet been investigated. In this work, phosphorothioate (PS) DNAs are evaluated in the synthesis of Au and Ag nanoclusters, and are employed to successfully template a novel emitter using T15 DNA at neutral pH. Mechanistic studies indicate a distinct UV-dependent formation mechanism that does not occur through the previously reported thymine N3. The positions of PS substitution have been optimized. This is the first reported use of a T15 template at physiological pH for AgNCs.

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.

Physical chemical characterization of DNA–SWNT suspensions and associated composites

Suspensions of DNA–SWNT composites have been prepared as part of an ongoing study of individualized single-walled carbon nanotubes (SWNTs) using chemically modified or unmodified single-stranded DNA as the surfactant. We report optical absorption, photoluminescence (PL) and Raman spectra which are used to distinguish the types of carbon nanotubes (CNTs) present in the suspensions. Pyrenyl groups appended to the DNA affect the appearance of absorption and photoluminescence spectra. Furthermore, TEM has been used to characterize DNA–SWNT composites deposited on grids and flushed with a volatile aqueous buffer. Electron micrographs provide preliminary evidence for helical wrapping of single strand DNA (ssDNA) around individual CNTs and thin ropes of CNTs.

The Identification and Characterization of a G4-DNA Resolvase Activity

There is increasing evidence that four-stranded Hoogsteen-bonded DNA structures, G4-DNA, play an important role in cellular processes such as meiosis and recombination. The Hoogsteen-bonded G4-DNA is thermodynamically more stable than duplex DNA, and many guanine-rich genomic DNA sequences with the ability to form G4-DNA have been identified. A protein-dependent activity that resolves G4-DNA into single-stranded DNA has been identified in human placental tissue. The resolvase activity was purified from any apparent nuclease activity and is dependent on NTP hydrolysis and MgCl2. Resolvase activity is optimal with 5 mM MgCl2. The Vmax/Km of ATP is 0. 055%/min/microM, higher than the Vmax/Km of the other dNTPs. The products of the resolvase reaction are unmodified single-stranded DNA. The resolvase is not a duplex DNA helicase or a topoisomerase II activity and does not unwind Hoogsteen-bonded triplex DNA. Resolvase is a novel activity that unwinds stable G4-DNA structures using a dNTP-dependent mechanism producing unmodified single-stranded DNA. Potential in vivo roles for this G4-DNA resolvase activity are discussed.

Lability of monofunctional cis-platinum adducts: role of DNA double helix.

Recently, we have shown that the adduct formed in the reaction between the platinum-triamine complex cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl]2+ and one single-stranded oligonucleotide was stable but became labile as soon as the platinated oligonucleotide was paired with its complementary strand (Gaucheron et al. Proc. Natl. Acad. Sci. USA 88, 3516-3519 (1991)). To generalize this finding we have now studied large DNA fragments containing several adducts. The stability of the adducts within single-stranded DNA is demonstrated by absorption spectrophotometry and by replication mapping experiments. Several approaches are used to prove the lability of the adducts within double-stranded DNA. Replication mapping experiments reveal that an unmodified single-stranded DNA when mixed with double-stranded DNA modified by the platinum-triamine complex behaves as a single-stranded DNA modified by the triamine complex. After double-stranded DNA is modified by the platinum-triamine complex, intrastrand and interstrand cross-links are progressively formed during subsequent incubation as revealed by transcription mapping experiments and gel electrophoresis under denaturing conditions. Finally, replication mapping experiments show that the lability of the adducts within a double-stranded DNA depends upon the nature of the flanking nucleotide residues. All these results support the proposal that the DNA double helix acts as a catalyst in the reaction between DNA, cis-diamminedichloroplatinum(II) and N-methyl-2,7-diazapyrenium.

Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy: Evidence for multiple heterogeneous DNA Co-ordination

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATPγS (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATPγS, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATPγS the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the typtophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.