Labeled DNA Oligomers Research Articles

Toward Quantitative Nanothermometry Using Single-Molecule Counting.

Photothermal heating of nanoparticles has applications in nanomedicine, photocatalysis, photoelectrochemistry, and data storage, but accurate measurements of temperature at the nanoparticle surface are lacking. Here we demonstrate progress toward a super-resolution DNA nanothermometry technique capable of reporting the surface temperature on single plasmonic nanoparticles. Gold nanoparticles are functionalized with double-stranded DNA, and the extent of DNA denaturation under heating conditions serves as a reporter of temperature. Fluorescently labeled DNA oligomers are used to probe the denatured DNA through transient binding interactions. By counting the number of fluorescent binding events as a function of temperature, we reconstruct DNA melting curves that reproduce trends seen for solution-phase DNA. In addition, we demonstrate our ability to control the temperature of denaturation by changing the Na+ concentration and the base pair length of the double-stranded DNA on the nanoparticle surface. This degree of control allows us to select narrow temperature windows to probe, providing quantitative measurements of temperature at nanoscale surfaces.

Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

Microdroplet temperature calibration via thermal dissociation of quenched DNA oligomers

The development of microscale analytical techniques has created an increasing demand for reliable and accurate heating at the microscale. Here, we present a novel method for calibrating the temperature of microdroplets using quenched, fluorescently labeled DNA oligomers. Upon melting, the 3' fluorophore of the reporter oligomer separates from the 5' quencher of its reverse complement, creating a fluorescent signal recorded as a melting curve. The melting temperature for a given oligomer is determined with a conventional quantitative polymerase chain reaction (qPCR) instrument and used to calibrate the temperature within a microdroplet, with identical buffer concentrations, heated with an infrared laser. Since significant premelt fluorescence prevents the use of a conventional (single-term) sigmoid or logistic function to describe the melting curve, we present a three-term sigmoid model that provides a very good match to the asymmetric fluorescence melting curve with premelting. Using mixtures of three oligomers of different lengths, we fit multiple three-term sigmoids to obtain precise comparison of the microscale and macroscale fluorescence melting curves using "extrapolated two-state" melting temperatures.

Human Replication Protein a (RPA) Can Diffuse Along Single Stranded DNA

Replication Protein A (RPA) is a hetero-trimeric protein that plays critical roles in many cellular processes. The main function of RPA is to bind to single stranded (ss) DNA and to regulate its availability during DNA metabolic processes. RPA is also known to interact with an array of other proteins in DNA replication, repair and recombination processes in eukaryotic organisms. RPA binds to the transient ssDNA that forms during nearly all aspects of DNA metabolism and protects ssDNA from nucleases. Although RPA binds to ssDNA with a very high affinity, it must be dissociated from or redistributed along ssDNA during DNA replication. To probe this re-arrangement of RPA along ssDNA, ensemble and single molecule studies have been used as complementary techniques to investigate human RPA diffusion along fluorescently labeled ssDNA oligomers. The dynamics of human RPA (hRPA) along fluorescently labeled DNA oligomers was also studied with fluorophore - labeled hRPA. These experiments illustrate that hRPA can spontaneously re-arrange along ssDNA by diffusing while remaining tightly bound. In addition, hRPA can also transiently melt DNA hairpin structures by diffusing in from ssDNA that is adjacent to the DNA hairpin. This ability of hRPA to diffuse along ssDNA means that directional DNA motor proteins such as polymerases or translocases can push RPA and re-organize it along ssDNA. This diffusion property of RPA is shared with the bacterial analogue of RPA, the E. coli SSB protein that also has previously been shown to diffuse along ssDNA. (GM030498 (TML), GM098509 (RG), GM044721 (MSW)).

Effect of Thiazole Orange Doubly Labeled Thymidine on DNA Duplex Formation

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ~ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/. It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.

Revival of High-Order Fluorescence Correlation Analysis: Generalized Theory and Biochemical Applications

Analysis of high-order correlations in fluorescence fluctuation spectroscopy was developed in the late 1980s but since then has been replaced by alternative brightness analysis methods. However, high-order correlation has important advantages in many experiments. We present a new cumulant-based formalism of high-order correlation that greatly simplifies data analysis. The new formalism is used to derive general expressions for variance of high-order correlations that show good agreement with experiment in a model system of fluorescently labeled DNA oligomers. A simulation of binary systems in which both diffusion time and brightness are varied illustrates clearly that high-order analysis has better sensitivity to brightness than fluorescence correlation spectroscopy (FCS). These results have implications for analysis of isomerization reactions and dual-beam FCS with flow. We also demonstrate that high-order correlations can detect photobleaching in the observation volume. The application of this formalism to many FCS-based experiments allows more accurate analysis in addition to describing more molecular parameters.

Site-Specific Immobilization of DNA in Glass Microchannels via Photolithography

For the first time, a microchannel was photochemically patterned with a functional linker. This simple method was developed for the site-specific attachment of DNA via this linker onto silicon oxide surfaces (e.g., fused silica and borosilicate glass), both onto a flat surface and onto the inside of a fused silica microchannel. Sharp boundaries in the micrometer range between modified and unmodified zones were demonstrated by the attachment of fluorescently labeled DNA oligomers. Studies of repeated hybridization-dehybridization cycles revealed selective and reversible binding of cDNA strands at the explicit locations. On average, approximately 7 x 10(11) fluorescently labeled DNA molecules were hybridized per square centimeter. The modified surfaces were characterized with X-ray photoelectron spectroscopy, infrared microscopy, static contact angle measurements, confocal laser scanning microscopy, and fluorescence detection (to quantify the attachment of the fluorescently labeled DNA).

Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Protected 32P‐Labels in Deoxyribonucleotides: Investigation of Sequence Selectivity of DNA Photocleavage by Enediyne–, Fulvene–, and Acetylene–Lysine Conjugates

Finding a compromise: The sequence selectivity of DNA photocleavage by enediyne–, acetylene–, and fulvene–lysine conjugates was investigated by using labeled DNA oligomers. Since the external 32P label was removed by these photocleavers, the label was protected by translocation inside the oligomer (see figure). The selectivity of cleavage is controlled by a compromise between AT selectivity for binding and G selectivity for electron transfer.

DNA Attachment Chemistry at the Flexible Silicone Elastomer Surface: Toward Disposable Microarrays

This paper describes the preparation and surface characterization of maleimide-activated silicone elastomer (PDMS(MCC)) followed by covalent functionalization using thiol-terminated DNA sequences (primary oligo). The stability of this attachment chemistry was demonstrated by the retention of the primary oligo through the process of hybridization with a labeled complementary DNA sequence. In these studies, the hybridized labeled DNA oligomers were detected using confocal fluorescence microscopy. We have employed a vapor deposition technique in which a plasma-treated silicone elastomer (PDMS(OH)) was exposed to vapors of 3-(aminopropyl)triethoxysilane (APTS) under vacuum, to yield the amine-functionalized silicone elastomer (PDMS(NH)(2)). PDMS(NH)(2) was further coupled with a heterofunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to obtain PDMS(MCC). The surface functionalities of the elastomers were characterized using contact angle measurements and X-ray photoelectron spectroscopy. Surface-modified silicone elastomers appear to be promising substrates for use as substrates for disposable microarrays.

Enhanced emission of highly labeled DNA oligomers near silver metallic surfaces.

Fluorescein is a widely used fluorescent probe in DNA analysis. One difficulty with fluorescein is its self-quenching due to resonance energy transfer between the residues, which results in decreased intensities with increasing labeling density. We examined the emission spectral properties of DNA oligomers labeled with one or five fluorescein residues. The emission intensity of the more highly labeled oligomer was decreased due to self-quenching. The self-quenching was mostly eliminated when this oligomer was held approximately 90 A from the surface of metallic silver particles. The intensities increased 7- and 19-fold for the oligomers with one or five fluoresceins, respectively. The increased intensity did not result in increased photobleaching. These results suggest the use of substrates coated with silver particles for increased sensitivity on DNA arrays or for DNA analysis.

Increased intensities of YOYO-1-labeled DNA oligomers near silver particles.

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.

Solid-phase synthesis of selectively labeled DNA: Applications for multidimensional nuclear magnetic resonance spectroscopy

The solid-phase chemical synthesis method has a strong advantage over the enzymatic method for preparing selectively labeled DNA oligomers. Atom-specific and fully labeled 2'-deoxynucleosides are economically prepared with routinely available isotope precursors using this synthetic route. Special DNA oligomers prepared by advanced labeling techniques are needed for advanced NMR applications, and chemical synthesis is the method of choice to respond to such demands. As a summary of this chapter, two tables are given. Table I lists the labeled nucleosides reported to be available by chemical syntheses. Table II lists the NMR studies using labeled DNA oligomers that were prepared by chemical syntheses.

Studies of physicochemical properties of N-H...N hydrogen bonds in DNA, using selective 15N-labeling and direct 15N 1D NMR.

15N-15N scalar coupling constants across base pair hydrogen bonds (2hJ(NN)) were studied using residue- and atom-specifically 15N labeled DNA oligomers. The N3 atom selectively 15N enriched 2'-deoxycytidine and thymidine, and the uniformly 15N enriched 2'-deoxyadenosine and 2'-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAATTCGCG)2 and d(CGCAAAAAGCG).d(CGCTTTTTGCG). This isotope labeling enabled us to determine the 2hJ(NN) value from the splitting of the 15N 1D spectrum. Additionally, it enabled the determination of 2hJ(NN) in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependence were examined for these oligomers. The sequence dependence was not clear; however, a significant decrease of 2hJ(NN) was observed by elevating the temperature. This temperature dependence was not due to the hydrogen exchange, since the addition of 20 mM NH3 did not change the 2hJ(NN) values. The 2hJ(NN) values in D2O were somewhat smaller than those in H2O. As compared to our 15N 1D method, the quantitative HNN-COSY method gave systematically smaller 2hJ(NN) values in our system, due to the lower 15N fraction of our sample (79 and 88% for dA and the other nucleotides, respectively) and the insufficient power of the 15N RF pulse (B1 = 6.6 kHz). These systematic differences were recovered by theoretical correction of the 15N isotope fraction contribution, by using the composite 15N 180 degrees pulse in a quantitative HNN-COSY experiment.

Synthesis, Chromatographic Separation, and Characterization of Near-Infrared Labeled DNA Oligomers for Use in DNA Sequencing

Traditionally, visible fluorophores have been used as labels in DNA sequencing. They absorb and fluoresce in a region of the spectrum that is susceptible to biological interferences in sequencing samples. The increased noise level due to autofluorescence of glass, solvent, or impurities can greatly reduce the potential sensitivity of the analysis. In an attempt to increase the sensitivity, we have investigated the use of near-infrared (near-IR) fluorophores as labels in DNA sequencing. Near-IR fluorophores possess spectral properties which are observed between 700 and 1200 nm, a region with characteristically little interference by biomolecules. Therefore, the detection of near-IR dyes is not limited by noise levels, but rather by detector capabilities. Near-IR dyes are also suitable for selective excitation with commercially available laser diodes which can further enhance the observed fluorescence signal. We have covalently linked fuction-alized near-IR dyes to moiled DNA oligomers for use in DNA sequencing. We report the synthesis and chromatographic purification of near-IR-labeled DNA oligomers. Wefurther discuss the inherent properties of the conjugates and their use in DNA sequencing procedures