Thymine Oligonucleotides Research Articles

Using a fluorescence quenching method to detect DNA adsorption onto single-walled carbon nanotube surfaces

Surface modification of single-walled carbon nanotubes (SWNTs) with DNA molecules has attracted much attention in recent years to increase SWNT solubility and make various SWNT-based nanobiodevices. Therefore, there is a critical need to quantify the interaction between DNA molecules and SWNT surfaces, particularly the intermediate structures during DNA adsorption. In this study, we demonstrate the ability to detect the adsorption of DNA oligomers on SWNT surfaces by fluorescence spectroscopy. Fluorescein-labelled, 30mer, thymine oligonucleotides (F-T30) were employed as a fluorescent probe to study the interaction of DNA with SWNTs. A clear quenching effect was observed when F-T30 was adsorbed onto SWNT surfaces. Using this method, the amount of DNA adsorbed onto the SWNT surfaces was measured under different sonication conditions to correlate adsorption efficiency with sonication strength and duration. When a bath-type sonicator was used, mild adsorption of F-T30 on SWNT surfaces was observed. Furthermore, a two-step adsorption was observed in this condition. In contrast, we observed rapid adsorption of F-T30 to SWNT surfaces at the higher sonication amplitude (60% maximal) using a probe-type sonicator, while only slight adsorption of DNA molecules was observed at the lower amplitude (20% maximal). Our data revealed that the quenching effect can be used to evaluate DNA adsorption onto SWNT surfaces. In addition, atomic force microscopy (AFM) and photoacoustic spectroscopy (PAS) were conducted to provide complementary information on the DNA-SWNT nanoconjugates.

Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

Counting the Number of Magnesium Ions Bound to the Surface-Immobilized Thymine Oligonucleotides That Comprise Spherical Nucleic Acids

Label-free studies carried out under aqueous phase conditions quantify the number of Mg(2+) ions binding to surface-immobilized T40 sequences, the subsequent reordering of DNA on the surface, and the consequences of Mg(2+) binding for DNA-DNA interactions. Second harmonic generation measurements indicate that, within error, 18-20 Mg(2+) ions are bound to the T40 strand at saturation and that the metal-DNA interaction is associated with a near 30% length contraction of the strand. Structural reordering, evaluated using vibrational sum frequency generation, atomic force microscopy, and dynamic light scattering, is attributed to increased charge screening as the Mg(2+) ions bind to the negatively charged DNA, reducing repulsive Coulomb forces between nucleotides and allowing the DNA single strands to collapse or coil upon themselves. The impact of Mg(2+) binding on DNA hybridization and duplex stability is assessed with spherical nucleic acid (SNA) gold nanoparticle conjugates in order to determine an optimal working range of Mg(2+) concentrations for DNA-DNA interactions in the absence of NaCl. The findings are consistent with a charge titration effect in which, in the absence of NaCl, (1) hybridization does not occur at room temperature if an average of 17.5 or less Mg(2+) ions are bound per T40 strand, which is not reached until the bulk Mg(2+) concentration approaches 0.5 mM; (2) hybridization proceeds, albeit with low duplex stability having an average Tm of 31(3)°C, if an average of 17.5-18.0 Mg(2+) ions are bound; and (3) highly stable duplexes having a Tm of 64(2)°C form if 18.5-19.0 Mg(2+) ions are bound, corresponding to saturation of the T40 strand.

Nano-Engineered Optimal Templates for Surface-Plasmon Enhanced Raman Scattering

We investigated the critical conditions to realize reliable and nano-engineered templates for surface-plasmon enhanced Raman scattering (SERS). Ultra-sensitive SERSs of thymine oligonucleotides were successfully realized on the template of Au nanoparticle arrays which were prepared by the combination of electron-beam lithography and post-chemical modification techniques. Drastic enhancement of Raman signal from the thymine oligonucleotides was only observed on the optimized templates, where the tuning of the plasmon resonance condition and the formation of the hot spots were both critical. Our results suggest that the artificial generation of reproducible and controlled hot spots can be achieved by our approach.

Metastable fragmentation of a thymidine-nucleotide and its components

In aqueous biological environment DNA is typically deprotonated. The metastable deprotonated DNA nucleobases are important reaction intermediates in low-energy electron induced damage to DNA. By comparing the metastable decay of small deprotonated DNA building blocks (thymine, thymidine, d-ribose, d-ribose 5-monophosphate) with the metastable decay of thymidine 5′-monophosphate and the hexameric oligonucleotide dT6 we show that the most intense fragmentation pathways of the individual components are inherited to their next larger compositions of one step further in complexity, i.e., from thymine and ribose to thymidine, from thymidine and 2-deoxyribose 5-monophosphate to thymidine 5′-monophosphate, and from thymidine 5′-monophosphate to thymine oligonucleotides. However, fragmentation channels that are dominant in the smaller, principle building blocks such as d-ribose and thymidine do not necessarily prevail beyond their composition, i.e., in thymidine 5′-monophosphate and thymine oligonucleotides. The comparison of the metastable decay mass spectra of molecules of increasing complexity reveals detailed fragmentation mechanisms and shows that the individual fragmentation pathways are determined by the initial deprotonation sites.

Label free sub-picomole level DNA detection with Ag nanoparticle decorated Au nanotip arrays as surface enhanced Raman spectroscopy platform

Label free optical sensing of adenine and thymine oligonucleotides has been achieved at the sub-picomole level using self assembled silver nanoparticles (AgNPs) decorated gold nanotip (AuNT) arrays. The platform consisting of the AuNTs not only aids in efficient bio-immobilization, but also packs AgNPs in a three dimensional high surface area workspace, assisting in surface enhanced Raman scattering (SERS). The use of sub-10 nm AgNPs with optimum inter-particle distance ensures amplification of the chemically specific Raman signals of the adsorbed adenine, thymine, cytosine and guanine molecules in SERS experiments. High temporal stability of the Raman signals ensured reliable and repeatable DNA detection even after three weeks of ambient desk-top conservation. This facile architecture, being three dimensional and non-lithographic, differs from conventional SERS platforms.

Asymmetrical Diaromatic Guanidinium/2-Aminoimidazolinium Derivatives: Synthesis and DNA Affinity

In this paper we report the synthesis of three families of new amidine-based aromatic derivatives as potential DNA minor groove binding agents for the treatment of cancer. The preparation of monoguanidine, mono-2-aminoimidazoline, and asymmetric diphenylguanidine/2-aminoimidazoline derivatives (compounds 1a-c to 8a-c) is presented. The affinity of these substrates and of a family of mono- and bis-isoureas (previously prepared in Rozas' laboratory) for DNA was evaluated by means of DNA thermal denaturation measurements. In particular, compounds 2c, 5c, 6c, 7c, and 8c were found to bind strongly both to natural DNA and to adenine-thymine oligonucleotides, showing a preference for the adenine-thymine base pair sequences.

Characterization by Raman spectroscopy of conformational changes on guanine–cytosine and adenine–thymine oligonucleotides induced by aminooxy analogues of spermidine

AbstractThe interactions between aminooxy analogues of the biogenic polyamine spermidine and selected 15‐mer oligodeoxyribonucleotides with alternating purine–pyrimidine base sequences, adenine–thymine (AT) and guanine–cytosine (GC), have been studied using Raman spectroscopy. These analogues resulted from the substitution of the two terminal aminomethylene groups of spermidine, +NH3CH2, by an aminooxy one, H2NO. Since this biogenic polyamine has no centre of symmetry, there are two different isosteric molecules, which have been named AOEPUT and APAPA. However, the pKa of an aminooxy group (about 5) is not high enough to be protonated at neutral pH. Therefore, under physiological conditions, these analogues have a positive charge less than spermidine, which makes them suitable models to investigate the roles of both charge and structure in the polyamine–DNA interactions. Raman spectra of solutions containing different concentrations of the analogues, from 10 to 75 mM, and either a GC and an AT oligonucleotide, at a fixed concentration 60 mM (in phosphate), were recorded. The spectra demonstrated the existence of strong differences in the oligonucleotide‐analogue interactions depending on the base sequences. In the presence of the aminooxy analogues, a 15‐mer GC oligonucleotide adopted a highly ordered tertiary structure (aggregate state or Ψ‐DNA) and, at the same time, it changed its secondary structure (from B to Z‐DNA). Evidence of macromolecular transitions was not achieved from the Raman spectra of a 15‐mer AT oligonucleotide. However, the assignments of the Raman bands indicated that both H2NO groups of the aminooxy analogues and exocyclic NH2 groups of the oligonucleotides could act as preferential binding sites. However, different spectral features were observed between AOEPUT and APAPA in the presence of the oligonucleotides. This fact supports the idea that the two amino terminal groups of spermidine could have different roles in the interaction of this biogenic polyamine with DNA. Our results also confirm that the aminooxy analogues of biogenic polyamines could act as stabilizers of non‐viral transfection vectors, thus improving the efficiency of gene therapy protocols. Copyright © 2004 John Wiley & Sons, Ltd.

Blackbody infrared radiative dissociation of oligonucleotide anions

The dissociation kinetics of a series of doubly deprotonated oligonucleotide 7-mers [ d(A) 7 2−, d(AATTAAT) 2−, d(TTAATTA) 2−, and d(CCGGCCG) 2−] were measured using blackbody infrared radiative dissociation in a Fourier-transform mass spectrometer. The oligonucleotides dissociate first by cleavage at the glycosidic bond leading to the loss of a neutral nucleobase, followed by cleavage at the adjacent (5′) phosphodiester bond to produce structurally informative a-base and w type ions. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained for the loss of base. The measured Arrhenius parameters are dependent on the identity of the nucleobase. The process involving the loss of an adenine base from the dianions, d(A) 7 2−, d(AATTAAT) 2−, and d(TTAATTA) 2− has an average activation energy ( E a ) of ∼1.0 eV and a preexponential factor ( A) of 10 10 s −1. Both guanine and cytosine base loss occurs for d(CCGGCCG) 2−. The average Arrhenius parameters for the loss of cytosine and guanine are E a = 1.32 ± 0.03 eV and A = 10 13.3±0.3 s −1. No loss of thymine was observed for mixed adenine–thymine oligonucleotides. Neither base loss nor any other fragmentation reactions occur for d(T) 7 2− over a 600 s reaction delay at 207 °C, a temperature close to the upper limit accessible with our instrument. The Arrhenius parameters indicate that the preferred cleavage sites for mixed oligonucleotides of similar mass-to-charge ratio will be strongly dependent on the internal energy of the precursor ions. At low internal energies (effective temperatures below 475 K), loss of adenine and subsequent cleavage of the adjacent phosphoester bonds will dominate, whereas at higher energies, preferential cleavage at C and G residues will occur. The magnitude of the A factors ≤10 13 s −1 measured for the loss of the three nucleobases (A, G, and C) is indicative of an entropically neutral or disfavored process as the rate limiting step for this reaction.

Specificity of Cellular DNA-Binding Sites of Microbial Populations in a Florida Reservoir

The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [H]- or [P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nucleic acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments.

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.

Studies on the nucleotide arrangement in deoxyribonucleic acids VI. Pyrimidine nucleotide clusters: Frequency and distribution in several species of the AT-type☆

The pyrimidine oligonucleotide clusters released by the hydrolysis of DNA specimens from calf thymus, rye germ, and wheat germ have been studied. Sequences of the general structure (pyrimidine deoxyriboside)n(phosphorric acid residue)n+1 with n of 1–8, have been separated quantitatively and their distribution in the three DNA preparations has been compared. Clusters of 9 or more pyrimidines in a row account for 6–8 % of the pyrimidines. In all cases, the results deviate considerably from what would be expected from a random arrangement of clusters. Almost all oligonucleotide units, comprised in the fractions with n of 1–4, have been separated, characterized, and estimated. Whereas the totol abundance of the various oligonucleotide classes was rather similar in the three DNA specimens, which were all of the AT-type, calf-thymus DNA differed widely from the specimens from rye and wheat germ in the amounts of individual oligonucleotides isolated. Some pyrimidine oligonucleotides which could have been expected were not encountered, conspicuously those consisting of 4 or 5 cytosine units in a row, whereas the corresponding thymine oligonucleotides were observed invariably. Previous observations showing that in the plant nucleic acid 5-methylcytosine and cytosine do not share the same pattern of arrangement within the polymer have been confirmed. The bearing of some of the findings on the general problem of DNA structure is considered.

Studies on the nucleotide arrangement in deoxyribonucleic acids VI. Pyrimidine nucleotide clusters: Frequency and distribution in several species of the AT-type

The pyrimidine oligonucleotide clusters released by the hydrolysis of DNA specimens from calf thymus, rye germ, and wheat germ have been studied. Sequences of the general structure (pyrimidine deoxyriboside) n (phosphorric acid residue) n+1 with n of 1–8, have been separated quantitatively and their distribution in the three DNA preparations has been compared. Clusters of 9 or more pyrimidines in a row account for 6–8 % of the pyrimidines. In all cases, the results deviate considerably from what would be expected from a random arrangement of clusters. Almost all oligonucleotide units, comprised in the fractions with n of 1–4, have been separated, characterized, and estimated. Whereas the totol abundance of the various oligonucleotide classes was rather similar in the three DNA specimens, which were all of the AT-type, calf-thymus DNA differed widely from the specimens from rye and wheat germ in the amounts of individual oligonucleotides isolated. Some pyrimidine oligonucleotides which could have been expected were not encountered, conspicuously those consisting of 4 or 5 cytosine units in a row, whereas the corresponding thymine oligonucleotides were observed invariably. Previous observations showing that in the plant nucleic acid 5-methylcytosine and cytosine do not share the same pattern of arrangement within the polymer have been confirmed. The bearing of some of the findings on the general problem of DNA structure is considered.