T-rich Oligonucleotides Research Articles

Multicolor Hg2+ sensor based on the regulation of peroxidase activity of Fe/Co-MIL-88(NH2) and etching of gold nanobipyramids

The entry of Hg2+ into the human body can lead to serious damage to some organs and it is vital to construct some simple but sensitive Hg2+ assay methods. A multicolor sensor for Hg2+ was constructed depended on the inhibition of peroxidase activity of Fe/Co-MIL-88(NH2) mediated by DNA and color change caused by the etching of gold nanobipyramids (Au NBPs). The enzymatic activity of Fe/Co-MIL-88(NH2) can be inhibited by adsorbing the T-rich oligonucleotide sequence on its surface. Hg2+ can combine with thymine of the T-rich oligonucleotide sequence to constitute T-Hg2+-T complex, and cause the transforming of T-rich oligonucleotide sequence into three-dimensional structure and desorbed from Fe/Co-MIL-88(NH2) surface. Which results in the restoration of the peroxidase activity of Fe/Co-MIL-88(NH2), and then it catalyzes the 3,3′,5,5′-tetramethylbenzidine (TMB) to engender TMB2+, TMB2+ can etch Au NBPs, this accompanied with a peak shift of the UV–vis spectrum and recognizable color changes of the system. Semi-quantitative can be realized using the multicolor changes of the Au NBPs as the reading mode. Furthermore, quantitative sensing can be achieved by measuring the UV–vis peak shift of the system. The linear response range is 1.0 nM–10 µM, and the detection limit (LOD) is 0.28 nM.

Intramolecular Folding of PolyT Oligonucleotides Induced by Cooperative Binding of Silver(I) Ions.

Ag+-bridged T-Ag+-T was recently discovered in a Ag+-DNA nanowire crystal, but it was reported that Ag+ had little to no affinity to T nucleobases and T-rich oligonucleotides in solution. Therefore, the binding mode for the formation of this type of novel metallo base pair in solution is elusive. Herein, we demonstrate that Ag+ can interact with polyT oligonucleotides once the concentration of Ag+ in solution exceeds a threshold value. The threshold value is independent of the concentration of the polyT oligonucleotide but is inversely proportional to the length of the polyT oligonucleotide. The polyT oligonucleotides are intramolecularly folded due to their positively cooperative formation and the stack of T-Ag+-T base pairs, resulting in the 5'- and 3'-ends being in close proximity to each other. The intramolecular Ag+-folded polyT oligonucleotide has a higher thermal stability than the duplex and can be reversibly modulated by cysteine.

An Ultrasensitive Gold Nanoband Aptasensor for Mercury(II) Detection in Aquatic Environment

Heavy metal contamination in aquatic environment has made great adverse impacts on human health and environment. In this work, a label-free electrochemical gold nanoband aptasensor (GNA) was developed using thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry for Hg2+ detection in drinking water. The gold nanoband sensor contains gold nanoband working electrode with a band thickness of 100 nm. The nanoscale working electrode increases the mass transfer rate and sensitivity for Hg2+ detection. Moreover, the gold nanoband sensors are cost-effective and can be easily regenerated by cutting the edge after the performance decreases dramatically compared with microelectrodes and disk electrodes. T-rich oligonucleotides modified with thiol group was self-assembled onto the working electrode through Au-S covalent bonding. In the presence of Hg2+, oligonucleotides will capture Hg2+, thus inducing the conformational change from single-strand to duplex-like structure that would promote electron transfer. Electrochemical impedance spectroscopy (EIS) was used to detect the Hg2+-mediated conformational changes. By optimizing experimental conditions, the linear range of proposed biosensor is from 0.1 nM to 1 μM, and the limit of detection is 40 pM (8 ppt). Furthermore, this nanobiosensors exhibited high selectivity and a great potential to detect trace Hg2+ in real samples from aquatic environment.

Titanocene binding to oligonucleotides

The binding of titanocene to DNA and RNA was examined by means of electrospray mass spectrometry. Titanocene served as a model for its therapeutically active derivatives. The binding preferences were probed by competition experiments with oligonucleotides of varying nucleobase compositions and sequences. Results from competition experiments revealed a generally increased preference for the binding to phosphate groups adjacent to thymidines, which is affected by the nucleobase sequence of T-rich oligonucleotides. More detailed information about the binding sites was obtained from tandem mass spectrometric experiments. The binding of the transition metal coordination center significantly altered the fragment ion patterns of the oligonucleotides. RNA was found to be less prone to adduct formation, due to intramolecular interactions. The findings from experiments on DNA and RNA were complemented by the examination of backbone- and ribose-modified oligonucleotides.

A Selective Surface-Enhanced Raman Scattering Sensor for Mercury(II) Based on a Porous Polymer Material and the Target-Mediated Displacement of a T-Rich Strand

A sensitive and selective surface-enhanced Raman scattering (SERS) sensor for mercury(II) was fabricated based on the target-mediated displacement of a T-rich oligonucleotide strand. A DNA/aptamer duplex was prepared by the hybridization between a tetramethylrhodamine(TMR)-labeled thymine(T)-rich Hg2+-specific aptamer (denoted as TMR-aptamer) and a thiolated adenine-rich capturing DNA. The duplex can be immobilized onto the SERS substrate of the Ag-moiety modified glycidyl methacrylate-ethylene dimethacrylate (denoted as Ag-GMA-EDMA) via self-assembly by the thiol anchor, in which the TMR-aptamer exists in a double-stranded chain. In this case, the label of the TMR moiety approaches the substrate surface and produces a strong SERS signal. Upon the addition of the target, a pair of TMR-aptamers could cooperatively coordinate with Hg2+ to form a stable duplex-like structure mediated by the T–Hg2+–T complex between two adjacent strands, which triggers the release of the TMR-aptamer from the SERS substrate surface, thus drawing the TMR tags away from the substrate with a significant decrease in the SERS signal. This optical sensor shows a sensitive response to Hg2+ in a concentration from 5 nM to 2.0 μM with a detection limit of 2.5 nM. The prepared sensor is negligibly responsive to other metal ions, can be easily regenerated, and shows good performance in real sample analysis.

Target-Induced DNA Mismatch for One-Spot Simultaneous Electrochemical Detection of Multiple Heavy Metals Based on the Amplification of DNA Concatemers

Here, we develop a sensitive and selective method for simultaneous detection of Ag+ and Hg2+ by target-induced conformational switch and DNA concatemers amplification protocol. Two dsDNA strands, one is thiol labeled C-rich oligonucleotide (C1) and its partially complementary DNA (P1), the other is thiol labeled T-rich oligonucleotide (C2) and its partially complementary DNA (P2), were firstly coimmobilized on electrode. When four auxiliary DNA strands, anthraquinone-2-carboxylic acid (AQ) labeled LS1 and LS2, thionine (Thi) labeled LS3 and LS4, were introduced, extended dsDNA polymers with lots of AQ moieties and Thi moieties could be formed on electrode, which were employed P1 and P2 as primers for concatemer reaction. In the presence of Ag+ and Hg2+, C1 and C2 on electrode via the insertion approach can fold into hairpin structures, resulting in the release of redox-tagged DNA concatemers from electrode with a substantially decreased redox current for quantitatively detecting the targets. As a result, the proposed aptasensor showed a good linear relationship between reduction peak currents and analyte concentrations in the ranges from 0.01 nM to 5 μM with a detection limit of 2 pM for Ag+, and 0.5 nM to 50 μM with a detection limit of 7.5 pM for Hg2+, respectively.

One-step fabrication of electrochemical biosensor based on DNA-modified three-dimensional reduced graphene oxide and chitosan nanocomposite for highly sensitive detection of Hg(II)

A novel DNA-modified nanocomposite of three-dimensional reduced graphene oxide and chitosan particles (CS@3D-rGO@DNA) was prepared by one-step method and used as a new electrochemical biosensing strategy to detect Hg2+ in drinking water. This biosensor was fabricated by the chemical coordination of T-Hg2+-T between the T-rich oligonucleotide strands contained in the as-prepared CS@3D-rGO@DNA nanocomposite and Hg2+, thereby varying the electrochemical signals. The developed electrochemical biosensor exhibited high sensitivity toward the determination of Hg2+ with a low detection limit of 0.016nM, ranging from 0.1nM to 10nM. This novel biosensor also showed high selectivity, good stability, and repeatability. Hence, this proposed CS@3D-rGO@DNA-based electrochemical strategy is feasible and may offer a simple, rapid, cost-effective, highly selective, and sensitive method for Hg2+ quantification.

Dual-channel detection of metallothioneins and mercury based on a mercury-mediated aptamer beacon using thymidine–mercury–thymidine complex as a quencher

A novel dual-channel strategy for the detection of metallothioneins (MTs) and Hg2+ has been developed based on a mercury-mediated aptamer beacon (MAB) using thymidine–mercury–thymidine complex as a quencher for the first time. In the presence of Hg2+, the T-rich oligonucleotide with a 6-carboxyfluorescein (TRO-FAM) can form an aptamer beacon via the formation of T–Hg2+–T base pairs, which results in a fluorescence quenching of the sensing system owing to the fluorescence resonance energy transfer (FRET) from the fluorophore of FAM to the terminated T–Hg2+–T base pair. The addition of MTs into this solution leads to the disruption of the T–Hg2+–T complex, resulting in an increase of the fluorescent signal of the system. In the optimizing condition, ΔF was directly proportional to the concentrations ranging from 5.63nM to 0.275μM for MTs, and 14.2nM to 0.30μM for Hg2+ with the detection limits of 1.69nM and 4.28nM, respectively. The proposed dual-channel method avoids the label steps of a quencher in common molecular beacon strategies, without tedious procedure or the requirement of sophisticated equipment, and is rapid, inexpensive and sensitive.

N-doped TiO2 based visible light activated label-free photoelectrochemical biosensor for detection of Hg(2+) through quenching of photogenerated electrons.

A novel photoelectrochemical (PEC) biosensor was fabricated based on N-doped TiO2 for the detection of Hg(2+) through the quenching of photogenerated electrons. The N-doped TiO2 was synthesized by a sol-gel method with urea and tetrabutyl titanate as the N and Ti sources. Compared with the undoped TiO2, the N-doped TiO2 showed an enhanced photocurrent response under visible light (λ > 420 nm). The sensing surface was functionalized with 5'-amino-modified T-rich oligonucleotides. The photoelectrochemical biosensor bound Hg(2+) on the surface by a highly specific T-Hg(2+)-T recognition. Hg(2+) on the surface of the N-doped TiO2 film withdrew the photogenerated electrons and decreased the recorded current signal. The dynamic linear range for Hg(2+) has been determined to be as low as 2-6 μM.

Biomagnetic separation and pre-concentration of trace amounts of Hg2+ in biological samples based on T-rich oligonucleotide modified magnetic beads

We report a simple and sensitive method for the analysis of trace mercury(ii) (Hg2+) in biological samples, based on a magnetic bead (MB) separation/pre-concentration process and thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry.

Colorimetric detection of metallothioneins using a thymine-rich oligonucleotide–Hg complex and gold nanoparticles

A simple and sensitive method for label-free, colorimetric detection of metallothioneins (MTs) has been developed by using a thymine (T)-rich oligonucleotide (TRO)–Hg–AuNP system. In this colorimetric strategy, the thiol groups of MTs could interact with mercury from the T–Hg2+–T complex to release TRO, resulting in a color change of the system. The response signals linearly correlated with the concentration of MTs over the range of 2.56×10−8 to 3.08×10−7molL−1, and the limit of detection was 7.67×10−9molL−1. The relative standard deviation and the recovery were 2.3–4.8% (n=11) and 94.2–103.9%, respectively. The proposed method avoids the label and derivatization steps in common methods, allows direct analysis of the samples by the naked eye without costly instruments, and is reliable, inexpensive, and sensitive.

Dual polarisation interferometry for real-time, label-free detection of interaction of mercury(ii) with mercury-specific oligonucleotides

A real-time, label-free dual polarisation interferometry technique was used to investigate the interaction of Hg(2+) with a 21-mer T-rich oligonucleotide and further construct a Hg(2+) biosensor based on thymine-Hg(2+)-thymine coordination chemistry.

Characterization of Activation Energy Distribution for Ultraviolet Irradiation of T-Rich Oligonucleotide Using High-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry Tandem Mass Spectrometry

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer of human being. Series of experiments were carried out in order to reveal activation energy distribution of DNA mutation caused by UV radiation. The T-rich oligonucleotides were exposed to UV radiation with increasing intensity for different durations. Photoproducts of T-rich oligonucleotide were investigated using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Two photoproducts of T-rich oligonucleotide were cis-syn cyclobutane pyrimidine dimmer (T[c,s]T) and the pyrimidine(6,4)pyrimidone product (T[6,4]T). Activation energy distribution of DNA mutation was calculated using a commercial kinetics analysis programs by Robert L. Braun and Alan K. Burnham , Lawrance Livermore International Laboratory (version 2.4.1). To use the software for deriving the kinetics parameters, the factor T (temperature) in the software was substituted with k1R, in which k1 is a factor, R is radiation intensity. The activation energy derived ranges from 55 to 110 kJ mol(-1). By the same software, those kinetics parameters were extrapolated to natural UV radiation process to predict DNA damage degree without the DNA repair process.

Thermal Decomposition of Multiply Charged T-Rich Oligonucleotide Anions in the Gas Phase. Influence of Internal Solvation on the Arrhenius Parameters for Neutral Base Loss

Arrhenius activation parameters (E(a), A) for the loss of neutral nucleobases from a series of T-rich, doubly and triply deprotonated 15- and 20-mer oligodeoxynucleotides (ODN) containing a single reactive base (X = A or C) with the sequence, XT14, XT19 and T19X, have been determined using the blackbody infrared radiative dissociation technique. The A-containing anions are significantly more reactive (> or =3000 times) than the C-containing ions over the temperature range investigated. Importantly, the Arrhenius parameters for the loss of AH exhibit a strong dependence on size of the ODN and, to some extent, the charge state; the Arrhenius parameters increase with size and charge (Ea = 29-39 kcal mol(-1), A = 10(15)-10(20) s(-1)). In contrast, the parameters for the loss of CH are much less sensitive to size (Ea = 35-39 kcal mol(-1), A = 10(14)-10(17) s(-1)). The results are consistent with a greater contribution from the internal solvation of the reactive base to the Arrhenius parameters for the loss of A, compared with C, from the 15- and 20-mers. To further probe differences in internal solvation of A and C, hydrogen/deuterium exchange was carried out on AT19(-3), T19A(-3), CT19(-3) and T19C(-3) using D2O as the exchange reagent. However, the H/D exchange results did not reveal any differences in internal solvation within the ODN anions. Arrhenius parameters for the dissociation of noncovalent complexes of T20(-3) and the neutral nucleobase AH or CH have also been determined. Differences in the parameters indicate differences in the nature of the intermolecular interactions. It is proposed that neutral A-T interactions (i.e., base-base), which originate in solution, dominate in the case of (T20 + AH)(-3), while charge solvation, involving CH and a deprotonated phosphate group, is present for (T20 + CH)(-3).

Comparison of a + T-Rich Oligonucleotides with and without Self-Complementary Sequence Using Ion-Pair Reversed-Phase High-Performance Liquid Chromatography/Tandem Electrospray Ionization Mass Spectrometry

Both A+T-rich oligonucleotides with and without self-complementary sequences were analyzed using ion- pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) by tryethylammonium acetate (TEAA) and hexafluoroisopropanol (HFIP) buffer systems to study the characteristics of their retention behavior and electrospray ionization tandem mass spectrometry (ESI-MS/MS) response. The results indicated that the chain length had the same effect on the retention of A+T-rich oligonucleotides in both of TEAA and HFIP buffer systems but the sequence had a different impact on the retention in the two buffer systems. A+T- rich oligonucleotides with a self-complementary sequence were much shorter than those without in the TEAA buffer system whereas a slight difference was observed in the HFIP buffer system. Similar total ion current (TIC) intensity was observed both in oligonucleotides with or without self-complementary sequence. The opposite trend of a change in the TIC intensities with increasing chain length were observed in both the TEAA and HFIP buffer systems. A lower charge state was predominant in the TEAA buffer system whereas a higher charge state was mainly distributed in the HFIP buffer system. The oligonucleotides without self-complementary sequences had a higher charge state than those with self-complementary sequences. A- and T- are more esily formed at a higher charge state whereas the sequence fragments will be formed more easily at a lower charge state in both A+T-rich oligonucleotides with and without self-complementary sequences.

Characterization of G-rich and T-Rich Oligonucleotides Using Ion-Pair Reversed-Phase High-Performance Liquid Chromatography/Tandem Electrospray Ionization Mass Spectrometry

Characteristics of G-rich and T-rich oligonucleotides were investigated to compare their retention time, total ion current (TIC) intensity, charge-state distribution and product ion using ion-pair reversed-phase high- performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Three commonly used mobile phases for the analysis of oligonucleotides, triethylammonium acetate (TEAA), triethylammonium bicarbonate (TEAB) and triethylammonium hexafluoroisopropanol (HFIP) have been utilized. Retention time of G-rich and T-rich oligonucleotides was significantly different in TEAA and TEAB buffer systems, while in the HFIP buffer system it was affected more by the length of oligonucleotides. On the other hand, the ESI-MS ion abundance in the HFIP buffer system was higher than that in both TEAA and TEAB buffers. The TIC intensity of T-rich oligonucleotides was much higher than that of G-rich oligonucleotides in all mobile phases. In addition, much higher charge-state fragments were observed in HFIP buffer system than that in the case of TEAA and TEAB buffer systems. Product ions of both G-rich and T-rich oligonucleotides were affected by charge state of parent ions and collision energy.

Synthesis of C-branched spermine tethered oligo-DNA and the thermal stability of the duplexes and triplexes

The first synthesis of the new C-branched spermine derivative 17, as well as its ability to stabilise DNA duplexes and triplexes, are reported. The C-branched spermine block 17 was converted into the corresponding O-(2-cyanoethyl)-( N, N-diisopropyl)phosphoramidite block 18 for incorporation at the 5′-end of DNA. It was also coupled to the 2′ of ara-U through a phosphate bridge, leading to the partially protected 3′-hydroxy block 23, which was either converted to the O-(2-cyanoethyl)-( N, N-diisopropyl)phosphoramidite 24 or to the 3′-succinate block 25, leading to the synthesis of three model DNA 14-mers: 27 with tethered spermine at the 5′-end, 28 with tethered spermine at the middle of the DNA strand, and 29 with spermine at the 2′-end, using standard automated solid-phase chemistry and deprotection procedures. The T m measurements showed that, at low salt with Mg 2+ within the pH range of 5.5 – 7.6, the 5′-spermine-DNA conjugate 27 gives stabilised DNA triplexes with Δ h T ms of ∼+3° C and Δ c T ms of +5.5° to +8.5° C and the 2′-spermine-DNA conjugate 29 gives Δ h T ms of ∼+5° to +6.5° C and Δ c T ms of +7.5° to +9° C over the underivatised DNA counterpart 30. In contrast, the spermine block conjugated to the middle of the DNA as in 28 gave no triplex formation. Without Mg 2+, these short T-rich oligonucleotides ( 27, 29 & 30) gave triplex formation only above 0.4M NaCl. At 1.4M, the Δ h T ms for 27 and 29 over 30 were +9.5° C while the Δ c T ms were +6° to +7° C. Oligonucleotide conjugates 27 & 29 gave only weak duplex stabilisations with Δ h T ms of ≈+2° and ≈+1° C, respectively, over 30 at low salt with Mg 2+ within the pH range 5.5 – 7.6.