Solvolysis of 2'-deoxyribonucleosides and oligo-2'-deoxyribonucleotides in aqueous pyrrolidine or ammonia solutions.

To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2'-ß-D-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N6-methyladenine, N4-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2'-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.

Multi-frequency impedance sensing for detection and sizing of DNA fragments.

Electronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 µm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead). Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard.

The chromatographic behaviour of new double-labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity.

The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.

DNA microarray analysis using a smartphone to detect the BRCA-1 gene.

DNA microarrays are used to examine changes in gene expression of a large number of genes simultaneously by fluorescent labeling of complementary DNAs (cDNAs). The major bottleneck in implementing microarray technology in resource-limited settings lies in the detection instrument used for generating images of spotted oligonucleotides post-hybridization. While various methods such as a lateral flow assay have been presented to accomplish point-of-care disease detection, there is no simple and effective instrument available to gather spot images maintaining the standard microarray procedures. Nanotechnology based sensors connected with a portable smartphone readout system have the potential to be implemented in microarray technology. Here, we describe a portable fluorescence microarray based imaging system connected to a smartphone for detecting breast cancer gene expression (BRCA-1) from exon 11. This is based on the interactive binding of probe DNA to Cy3-target DNA. A paper-based microfluidics approach was used to demonstrate the DNA hybridization assay. The imaging principles of the assembled device named "FluoroZen" are similar to those of a fluorescence microscope. It uses two light spectrum filters, one to excite the fluorescent dye and the other to capture the emission spectrum. The images were acquired by using CCD cameras from FluoroZen. The smartphone integrated paper microfluidics platform presented here could be translated into clinical settings to perform point-of-care testing.

Exonuclease III-assisted multiple cycle amplification for the sensitive detection of DNA with zero background signal.

Detection of low-abundant DNA is essential for disease diagnosis and treatment. DNA polymerase-based amplification is frequently used due to its excellent sensitivity, but it suffers from time-consuming and labour-intensive procedures, complex template/primer design, and inherent nonspecific amplification. Alternatively, Exonuclease III (Exo III)-assisted target recycling provides a new approach for DNA assay because of its simplicity and general applicability, but it suffers from high background signal due to the nonspecific Exo III digestion and poor sensitivity due to single cycle signal amplification. Herein, we demonstrate the development of Exo III-assisted multiple cycle amplification (exonuclease chain reaction) for the sensitive detection of DNA with zero background signal. The binding of single-stranded DNA binding protein (SSB) to the hairpin probes can protect them from nonspecific digestion by Exo III, resulting in near zero background signal. The presence of the target DNA initiates the Exo III-triggered multiple cycle amplification, enabling the achievement of high sensitivity with a detection limit of 3 fM and excellent selectivity with single base mismatch discrimination capability, holding great potential in disease diagnosis and biomedical research.

Organic Semiconductor Laser Platform for the Detection of DNA by AgNP Plasmonic Enhancement.

Organic semiconductor lasers are a sensitive biosensing platform that respond to specific biomolecule binding events. So far, such biosensors have utilized protein-based interactions for surface functionalization but a nucleic acid-based strategy would considerably widen their utility as a general biodiagnostic platform. This manuscript reports two important advances for DNA-based sensing using an organic semiconductor (OS) distributed feedback (DFB) laser. First, the immobilization of alkyne-tagged 12/18-mer oligodeoxyribonucleotide (ODN) probes by Cu-catalyzed azide alkyne cycloaddition (CuAAC) or "click-chemistry" onto an 80 nm thick OS laser film modified with an azide-presenting polyelectrolyte monolayer is presented. Second, sequence-selective binding to these immobilized probes with complementary ODN-functionalized silver nanoparticles, is detected. As binding occurs, the nanoparticles increase the optical losses of the laser mode through plasmonic scattering and absorption, and this causes a rise in the threshold pump energy required for laser action that is proportional to the analyte concentration. By monitoring this threshold, detection of the complementary ODN target down to 11.5 pM is achieved. This complementary binding on the laser surface is independently confirmed through surface-enhanced Raman spectroscopy (SERS).

A domain-based DNA circuit for smart single-nucleotide variant identification.

According to the differential information of four homologous oligonucleotides, two domain-based encoders have been constructed with the molecular information as the input. Based on the one-to-one correspondence between the input and output, SNVs can be identified and their sites can be located at the domain level.

Quantitative surface-enhanced Raman spectroscopy of single bases in oligodeoxynucleotides.

To address the question of whether the SERS signals of ss-DNA are simply combinations of the signals from the individual bases that comprise the sequence, SERS spectra of unmodified ss-DNA sequences were obtained using a hydroxylamine-reduced Ag colloid aggregated with MgSO4. Initially, synthetic oligodeoxynucleotides with systematic structural variations were used to investigate the effect of adding single nucleobases to the 3' terminus of 10-mer and 20-mer sequences. It was found that the resulting SERS difference spectra could be used to identify the added nucleobases since they closely matched reference spectra of the same nucleobase. Investigation of the variation in intensity of an adenine probe which was moved along a test sequence showed there was a small end effect where nucleobases near the 3' terminus gave slightly larger signals but the effect was minor (30%). More significantly, in a sample set comprising 25-mer sequences where A, T or G nucleobases were substituted either near the centres of the sequences or the 5' or 3' ends, the SERS difference spectra only matched the expected form in approximately half the cases tested. This variation appeared to be due to changes in secondary structure induced by altering the sequences since uncoiling the sequences in a thermal pre-treatment step gave difference spectra which in all cases matched the expected form. Multivariate analysis of the set of substitution data showed that 99% of the variance could be accounted for in a model with just three factors whose loadings matched the spectra of the A, T, and G nucleobases and which contained no positional information. This suggests that aside from the differences in secondary structure which can be eliminated by thermal pre-treatment, the SERS spectra of the 25-mers studied here are simply the sum of their component parts. Although this means that SERS provides very little information on the primary sequence it should be excellent for the detection of post-transcription modifications to DNA which can occur at multiple positions along a given sequence.

Highly Improved Electrospray Ionization-Mass Spectrometry Detection of G-Quadruplex-Folded Oligonucleotides and Their Complexes with Small Molecules.

G-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases.

SERS and SERRS Detection of the DNA Lesion 8-Nitroguanine: A Self-Labeling Modification.

Rapid and sensitive methods to detect DNA lesions are essential in order to understand their role in carcinogenesis and for potential diagnosis of cancers. The 8-nitroguanine DNA lesion, which is closely associated with inflammation-induced cancers, has been characterized for the first time by surface-enhanced Raman spectroscopy (SERS). This lesion has been studied as the free base, as well as part of a dinucleotide and oligodeoxynucleotides (ODNs) at 5 different excitation wavelengths in the range 785-488 nm. All nitrated samples produced distinctly different spectra from their control guanine counterparts, with nitro bands being assigned by DFT calculations. Additional resonance enhancement was observed at the shorter excitation wavelengths, these SERRS measurements allowed the detection of one nitrated guanine in over 1,300 bases. In addition, SER(R)S can be used to detect whether the unstable lesion is covalently attached to the ODN or has been released by hydrolytic depurination.

Wide-Bore Columns of Poly(Glycidyl Methacrylate-Co-Divinylbenzene)-Based Monolithic Beds for Reversed-Phase and Anion-Exchange Chromatographic Separation of Biomolecules.

Three monoliths based on poly(glycidyl methacrylate-co-divinylbenzene) were prepared in the confines of borosilicate glass columns (100 × 3 mm I.D.). The first monolith was applied for analysis of proteins by reversed-phase high-performance liquid chromatography. It furnished a fast base-line separation for four proteins (ribonuclease A, cytochrome c, α-lactalbumin and myoglobin) in <80 s, with optimum resolution range of 2.11-2.84, and extremely small values of peak width at half height with a range of 1.0-1.6 s. The second and third monoliths were surface-modified into weak and strong anion-exchangers, respectively, and were investigated for anion-exchange (AE) high-performance liquid chromatography of four proteins with acidic isoelectronic points (bovine carbonic anhydrase, conalbumin, ovalbumin and soybean trypsin inhibitor) and of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)12-18]. The weak AE monolith experienced complete elution of the four proteins applying a basic Tris-HCl buffer (0.02 M, pH 8.9); however, the strong AE monolith established a base-line separation of these proteins in ~14 min. Both monoliths showed base-line separation of the seven fragments of d(pT)12-18 in ~6 min. The ion-exchange capacity determined by frontal and elemental analyses was comparable for the weak AE monolith (0.75 and 0.80 meq/g) and for the strong AE monolith (0.81 and 0.87 meq/g), respectively. Finally, a run-to-run and monolith-to-monolith reproducibility showed a relative standard deviation in retention time of d(pT)12-18 fragments of <2%.

Temperature-independent Detection of Heteroduplex and Homoduplex Fragments Applying Poly(glycidylmethacryate-co-divinylbenzene-based) Monoliths Modified to Strong Anion-exchanger.

Monoliths of poly(glycidylmethacrylate-co-divinylbenzene) were prepared in the confines of presilanized borosilicate glass columns (100 × 3 mm I.D.). These monoliths were surface modified into strong anion-exchangers with hydrochloric acid (10%) and triethylamine, successively. The strong anion-exchanger established good separation of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)(12-18)]. Moreover, heteroduplex and homoduplex fragments of a low-range mutation standard [of STS marker from the Y-chromosome (209 bp)] were separated at ambient and elevated temperatures using sodium phosphate buffer and a gradient former of sodium chloride, in anion-exchange high-performance liquid chromatography (AE-HPLC). This is a step forward for mutation detection as temperature-independent method, which is not the case in denatured ion-paired reversed-phase chromatography (D-IP-RP-HPLC), where mutation detection is temperature critical and might be bypassed if temperature changes slightly. Finally, reproducibility check from run-to-run and monolith-to-monolith showed a relative standard deviation (RSD) of less than 2%.

Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

Denaturing reversed-phase HPLC using a mobile phase containing urea for oligodeoxynucleotide analysis.

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.

A novel combined bisulfite UDG assay for selective 5-methylcytosine detection.

DNA modification, a significant epigenetic event, largely affects genes' binding with the transcription factors and some other DNA binding proteins. Among DNA modifications, methylation, especially cytosine methylation is of great importance and attracts extensive studies as it leads to the silence of tumor-suppressor gene expression. In this work, a novel combined bisulfite Uracil-DNA glycosylase (UDG) assay has been developed on the basis of bisulfite modification to generate uracil from cytosine, subsequent UDG-mediated uracil elimination and ultimate DNA cleavage in alkaline condition. This strategy can be used to selectively detect exact number and loci of 5-methylcytosine residues regardless of sequence context. Moreover, it provides linear quantitative results of DNA methylation level across a wide range.

Using surface-assisted laser desorption/ionization mass spectrometry to detect ss- and ds-oligodeoxynucleotides.

We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg(2+) (7.5 µM), and cadaverine (10 µM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T15, T20, T33, and T40, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation <23 %. In the presence of Ag(+) (1 µM) and cadaverine (10 µM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the ß-globin of sickle cell megaloblasts.

Simultaneous detection of duplex DNA oligonucleotides using a SERS-based micro-network gradient chip.

We report the development of a programmable surface-enhanced Raman scattering (SERS)-based micro-network gradient platform to simultaneously detect two different types of DNA oligomer mixtures. The utility of this platform was demonstrated by quantitative analysis of two breast cancer-related (BRCA1) DNA oligomer mixtures. To generate on-demand concentration gradients, the microfluidic circuit was designed using an electric-hydraulic analogy. Then a multi-gradient microfluidic channel was fabricated based on the theoretical design of the concentration control module. These micro-network structures automatically produce a series of different concentration gradients by continuously mixing Cy3-labeled DNA oligomers (BRAC1-Mutation) with TAMRA-labeled DNA oligomer (BRAC1-Wild). The SERS signals for different ratios of duplex DNA oligomer mixtures, adsorbed on the surface of silver nanoparticles, were measured under flowing conditions. Total analysis time from serial mixing to SERS detection takes less than 10 min because all experimental conditions are automatically controlled inside the exquisitely designed microfluidic channel. This novel SERS-based DNA sensing technology in a micro-network gradient channel is expected to be a powerful analytical tool to simultaneously detect multiple DNA oligomer mixtures.

ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications.

The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples.

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified.

A label-free optical sensor based on nanoporous gold arrays for the detection of oligodeoxynucleotides.

Interest in using nanoporous materials for sensing applications has increased. The present study reports a method of preparing well-ordered nanoporous gold arrays using a porous silicon (PSi) template. Gold nanolayer could be electrodeposited on the surface of the PSi template at low electrolysis currents in low concentration of chloroauric acid (HAuCl(4)) solution. Surface morphology characterizations and optical measurements revealed that a PSi-templated nanoporous gold (Au-PSi) array well replicated the nanoporous structure and retained the optical properties of PSi. Fourier transform reflectometric interference spectra showed that a characteristic blue-shifted effective optical thickness (EOT) was observed due to the low refractive index of the gold film. An optical DNA biosensor was then fabricated via the self-assembly of single-stranded DNA (ssDNA) with a specific sequence on the surface of Au-PSi. The attachment of ssDNA and its hybridization with target oligonucleotides (ODNs) persistently caused the blue shift of the EOT. Consequently, a relationship between the EOT shift and the ODN concentration was established. The mechanism of the optical response caused by DNA hybridization on the Au-PSi surface was qualitatively explained by the electromagnetic theory and electrochemical impedance spectroscopy (EIS). The lowest detection limit for target ODNs was estimated at around 10(-14) mol L(-1), when the baseline noise, a variation in the value of EOT is around 5 nm. The fabricated Au-PSi based optical biosensor has potential use in the discovery of new ODN drugs because it will be able to detect the binding event between ODNs and the target DNA.