DNA Synthesizer Research Articles

Syntheses of DNA Duplexes That Contain a N4C‐Alkyl‐N4C Interstrand Cross‐Link

This unit describes a simple procedure for preparing short DNA duplexes that contain a single N(4)C-alkyl-N(4)C interstrand cross-link. The synthesis is carried out on an automated DNA synthesizer using standard phosphoramidite chemistry. The cross-link is introduced during the synthesis of the duplex. The method can be used to prepare milligram quantities of cross-linked duplexes suitable for physical studies and for the preparation of larger DNA molecules that can be used as substrates to study DNA repair in whole cell extracts and in living cells in culture.

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670nm. We developed a new approach to antibody conjugation, using Huisgen-Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.

Differentiating between Fluorescence-Quenching Metal Ions with Polyfluorophore Sensors Built on a DNA Backbone

A common problem in detecting metal ions with fluorescentchemosensors is the emission-suppressing effects of fluorescence-quenching metal ions. This quenching tendency makes it difficult to design sensors with turn-on signal, and differentiate between several metal ions that may yield a strong quenching response. To address these challenges, we investigate a new sensor design strategy, incorporating fluorophores and metal ligands as DNA base replacements in DNA-like oligomers, for generating a broader range of responses for quenching metal ions. The modular molecular design enabled rapid synthesis and discovery of sensors from libraries on PEG-polystyrene beads. Using this approach, water-soluble sensors 1-5 were identified as strong responders to a set of eight typically quenching metal ions (Co(2+), Ni(2+), Cu(2+), Hg(2+), Pb(2+), Ag(+), Cr(3+), and Fe(3+)). They were synthesized and characterized for sensing responses in solution. Cross-screening with the full set of metal ions showed that they have a wide variety of responses, including emission enhancements and red- and blue-shifts. The diversity of sensor responses allows as few as two sensors (1 and 2) to be used together to successfully differentiate these eight metals. As a test, a set of unknown metal ion solutions in blind studies were also successfully identified based on the response pattern of the sensors. The modular nature of the sensor design strategy suggests a broadly applicable approach to finding sensors for differentiating many different cations by pattern-based recognition, simply by varying the sequence and composition of ligands and fluorophores on a DNA synthesizer.

Nanocluster Beacon (NCB): A DNA-Silver Nanocluster Probe that Fluoresces upon Hybridization

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. Recently we discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when brought into close proximity to a guanine-rich DNA sequence (Yeh et al., Nano Letters, 10 (8): 3106-3110, 2010). Based on this finding, we developed a new type of molecular probe (termed NanoCluster Beacon, NCB) that fluoresces upon target DNA binding. Compared to molecular beacons, NCBs require only a single labeling step and do not rely on F [[Unable to Display Character: ő]]rster energy transfer as fluorescence switching mechanism. Moreover, there is no need to remove the silver nanocluster precursors (Ag+ and BH4-) used during nanocluster formation, as these are essentially non-fluorescent. Our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers. It is also the first demonstration that a turn-on probe can be made based on fluorescent noble metal nanoclusters.Our initial investigation demonstrated NCB detection of an influenza target with a signal-to-background (S/B) ratio five times better than that of a conventional molecular beacon. Here, we expand upon this work to demonstrate a method of using NCBs to differentiate single-nucleotide variations. Our method discriminates single-nucleotide variants by colorimetric change of the NCB probes rather than fluorescence intensity change. This added dimensionality enables the increase in fluorescence intensity (on/off switching) to quantify the amount of target, whereas the fluorescence color identifies single-nucleotide variants. Samples with single-nucleotide variations can be unambiguously identified on a common gel imager with naked eyes, making this method a reliable and low-cost assay with simple readout format.Ref: H.-C. Yeh et al., “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10 (8): 3106-3110, 2010.

Qualitative and Quantitative Determination of Oligonucleotides by Non-Gel Capillary Electrophoresis

A novel simple non-gel capillary electrophoresis (NGCE) method was developed for the qualitative and quantitative determination of oligonucleotides (ODNs). ODNs were synthesized in a DNA synthesizer and separated by NGCE under optimized conditions as follows: 25 mmol L−1 Tris–boric–EDTA buffer (pH 8.0) with 7 mmol L−1 urea in the presence of 20% (w/v) polyethylene glycol (PEG) 35000 at 30 °C and −20 kV. Single-base differences, in which there are differences in the base quantity and the single-base structure with the same base quantity, can be identified by the proposed method, and the quantitative determination was carried out. The obtained regression equation revealed a good linear relationship between the peak area ratio and the ODNs concentration. The correlation coefficient was 0.9986 and the relative standard deviation (RSD) of the precision was <3.6% (intro-day) and <6.2% (inter-day), respectively. The recoveries of three concentrations (high, middle, and low) were between 99.6% and 107.6%. The established method is sensitive, simple, economical, and can be used in gene mutation detection, pharmacokinetics study of nucleic acid drugs, and so on.

Chemical synthesis and improved expression of recombinant human granulocyte colony-stimulating factor cDNA

Recently, granulocyte colony-stimulating factor (G-CSF) has been recognized as a useful molecule for the treatment of a wide range of complex ailments, such as cancer, AIDS, H1N1 influenza, cardiac and neurological diseases. The vast therapeutic potential of G-CSF has induced scientists to develop biotechnological approaches for the synthesis of this pharmacologically active agent. We used a synthetic G-CSF cDNA molecule to produce the target protein by a simple cloning protocol. We constructed the synthetic cDNA using a DNA synthesizer with the aim to increase its expression level by specific sequence modifications at the 5' end of the G-CSF-coding region, decreasing the GC content without altering the predicted amino acid sequences. The identity of the resulting protein was confirmed by a highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, commercial utilization of this methodology will require rigorous validation and quality control.

Design, Syntheses and Properties of Nucleic Acid Switch in Response to External Stimuli

The control of molecular properties using external stimuli is an attractive research area that offers the potential for regulation of various biological phenomena. This review summarizes the concept, design, syntheses and properties of nucleic acid switches in response to external stimuli, namely, "external-stimuli-responsive bridged nucleic acids monomer". From (1)H-NMR experiments, every external-stimuli-responsive bridged nucleic acid monomer was found to have an N-conformation, while an S-conformation was predominantly observed after exposure to a proper stimulus. Each bridged nucleic acid monomer was effectively introduced into oligodeoxynucleotides using an automated DNA synthesizer. Moreover, oligonucleotides modified with these bridged nucleic acid monomer were changed in their hybridization property and tolerance to enzymatic digestion in response to each stimulus. These results clearly showed that external-stimuli-responsive bridged nucleic acids monomers should work as a nucleic acid switch, and have the potential for regulation of various biological phenomena.

Diamondoid-modified DNA

We prepared novel C5-modified triphosphates and phosphoramidites with a diamondoid functionally linked to the nucleobase. Using primer extension experiments with different length templates we investigated whether the modified triphosphates were enzymatically incorporated into DNA and whether they were further extended. We found that all three modified nucleotides can be incorporated into DNA using a single-nucleotide incorporation experiment, but only partially using two templates that demand for multiple incorporation of the modified nucleotides. The modified phosphoramidites were introduced into oligonucleotides utilizing DNA synthesizer technology. The occurring oligonucleotide structures were examined by circular dichroism (CD) and melting temperature (T(m)) measurements and were found to adapt similar helix conformations as their unmodified counterparts.

Nucleotides. Part LXXVIII

AbstractSeveral N(‐hydroxyalkyl)‐2,4‐dinitroanilines were transformed into their phosphoramidites (see 5 and 6 in Scheme 1) in view of their use as fluorescence quenchers, and modified 2‐aminobenzamides (see 9, 10, 18, and 19 in Scheme 1) were applied in model reactions as fluorophors to determine the relative fluorescence quantum yields of the 3′‐Aba and 5′‐Dnp‐3′‐Aba conjugates (Aba=aminobenzamide, Dnp=dinitroaniline). Thymidine was alkylated with N‐(2‐chloroethyl)‐2,4‐dinitroaniline (24) to give 25 which was further modified to the building blocks 27 and 28 (Scheme 3). The 2‐amino group in 29 was transformed by diazotation into the 2‐fluoroinosine derivative 30 used as starting material for several reactions at the pyrimidine nucleus (→31, 33, and 35; Scheme 4). The 3′,5′‐di‐O‐acetyl‐2′‐deoxy‐N2‐[(dimethylamino)methylene]guanosine (37) was alkylated with methyl and ethyl iodide preferentially at N(1) to 43 and 44, and similarly reacted N‐(2‐chloroethyl)‐2,4‐dinitroaniline (24) to 38 and the N‐(2‐iodoethyl)‐N‐methyl analog 50 to 53 (Scheme 5). The 2′‐deoxyguanosine derivative 53 was transformed into 3′,5′‐di‐O‐acetyl‐2‐fluoro‐1‐{2‐[(2,4‐dinitrophenyl)methylamino]ethyl}inosine (54; Scheme 5) which reacted with 2,2′‐[ethane‐1,2‐diylbis(oxy)]bis[ethanamine] to modify the 2‐position with an amino spacer resulting in 56 (Scheme 6). Attachment of the fluorescein moiety 55 at 56 via a urea linkage led to the doubly labeled 2′‐deoxyguanosine derivative 57 (Scheme 6). Dimethoxytritylation to 58 and further reaction to the 3′‐succinate 59 and 3′‐phosphoramidite 60 afforded the common building blocks for the oligonucleotide synthesis (Scheme 6). Similarly, 30 reacted with N‐(2‐aminoethyl)‐2,4‐dinitroaniline (61) thus attaching the quencher at the 2‐position to yield 62 (Scheme 7). The amino spacer was again attached at the same site via a urea bridge to form 64. The labeling of 64 with the fluorescein derivative 55 was straigthforward giving 65. and dimethoxytritylation to 66 and further phosphitylation to 67 followed known procedures (Scheme 7). Several oligo‐2′‐deoxynucleotides containing the doubly labeled 2′‐deoxyguanosines at various positions of the chain were formed in a DNA synthesizer, and their fluorescence properties and the Tms in comparison to their parent duplexes were measured (Tables 1–5).

Synthesis and Properties of Fluorescent Biological Molecules Labeled with Novel Silylated Perylene Derivative

We have synthesized novel perylene derivatives bearing a silyl function with modifiable functional groups as a feasible material for visualizing biological substances. The fluorescent perylene derivative was coupled to C-5 position of pyrimidine nucleotide and the nucleotide was subsequently incorporated to the middle position of oligoDNA using an automated DNA synthesizer. Also, the derivative was coupled with a cholesterol analog with different length of linking alkyl function. The resulting modified fluorescent oligoDNA exhibited a small change in its fluorescent signal upon hybridization with the complementary oligoDNA. While the cholesterol analogs bearing the silylated perylene exhibited marked fluorescent signal in living cells. The signal is stronger in the analog having longer linker function compared to the analog having shorter linker function. In each case, however, the signal was much prominent compared to that of dehydroergosterol (DHE), a fluorescent cholesterol analog widely used in biological study.

A DNA−Silver Nanocluster Probe That Fluoresces upon Hybridization

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that "lights up" upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.

High‐Density DNA Functionalization by a Combination of Cu‐Catalyzed and Cu‐Free Click Chemistry

We report the regioselective Cu-free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3-Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5-disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu-free and Cu-catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high-density functionalization of DNA is simple, modular, and efficient.

ChemInform Abstract: Syntheses and Properties of Conformationally Restrained Nucleosides and Oligonucleotides Analogues

This review summarizes our efficient syntheses of novel bicyclic nucleoside analogues, 3'-O,4'-C-methyleneribonucleosides (1) (4-BC type nucleoside analogue), 2'-O,4'-C-methyleneribonucleosides (2) (5-BC), 3'-amino-3'-deoxy-3'-N,4'-C-methyleneribonucleosides (3) (aza 4-BC), and 3'-azido- and 3'-amino-3'-deoxy-2'-O,4'-C-methyleneribonucleosides (4, 5) (aza 5-BC). From 1H-NMR and X-ray crystallographic analyses, the 4-BC and aza 4-BC type nucleoside analogues (1, 3) were found to have a S-conformation predominantly, while the conformations of 5-BC and aza 5-BC type nucleoside analogues (2, 4, 5) were exclusively locked in N-form. The 4-BC and 5-BC type nucleoside analogues (1, 2) were effectively introduced into oligonucleotides using a DNA synthesizer. Furthermore, unprecedented hybridizing ability towards complementary RNA and DNA, RNA selectivity, potent triplex forming ability, and sufficient enzymatic stability of these modified oligonucleotides were also confirmed. These results should reveal a promising route to the development of antisense/antigene methodology.

Nucleotides. Part LXXVI

AbstractA series of new fluorescing 8‐(6‐hydroxyhexyl)isoalloxazine (=8‐(6‐hydroxyhexyl)benzo[g]pteridine‐2,4(1H,3H)‐dione) derivatives 4–13 were synthesized from 6‐[(6‐hydroxyhexyl)amino]uracil (2) with 1‐chloro‐4‐nitrosobenzene via 8‐chloro‐10‐(6‐hydroxyhexyl)isoalloxazine (3) and subsequent substitution of the Cl‐atom of 3 by various amines (Scheme). Analogously, 8‐substituted 10‐{3‐[(2,2‐dimethyl‐1,3‐dioxolan‐4‐yl)methoxy]propyl}isoalloxazines 19, 20, and 23–25 were prepared which yielded on deprotection the corresponding 10‐[3‐(2,3‐dihydroxypropoxy)propyl]alloxazines 21, 22, and 26–28. Their conversion into the 3″‐O‐(4,4′‐dimethoxytrityl) derivatives 29–33 and subsequent transformation into the corresponding 2″‐(2‐cyanoethyl N,N‐diisopropylphosphoramidites) 34–38 led to new building blocks for oligonucleotide synthesis. A series of 21‐mer oligodeoxyribonucleotides carrying the fluorescing isoalloxazine 37 in various positions of the chain were assembled in a DNA synthesizer. Combination with the complementary sequence yielded the stable duplexes 40–54 showing by the melting temperatures Tm that the fluorophor (F) does not harm the stability of the unmodified duplex 39 (Table).

In situ Synthesis of DNA Microarray on Functionalized Cyclic Olefin Copolymer Substrate

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.

Carbohydrate‐Oligonucleotide Conjugates

This unit describes a strategy for attaching two mannose and two galactose residues to an oligonucleotide. This conjugation can be performed at the 5'-end of the oligonucleotide sequence, using modified phosphoramidites. First, the oligonucleotide scaffold is synthesized on solid support using a DNA synthesizer, with commercially available and modified phosphoramidites. After the first "click" reaction with a galactosylated azide derivative on solid support, the bromine atoms are replaced with azides and a second click reaction is performed with propargylated mannose either on solid support or in solution. Additionally, using a monoalkynated solid support, the conjugation with carbohydrate residues can be performed at the 3'-end of the oligonucleotide according to a similar protocol. Curr. Protoc. Nucleic Acid Chem. 39:4.38.1-4.38.25.

Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.

DNA ligation using photoremovable protecting groups

Template-directed ligation of oligonucleotides by photochemical reaction has attracted much interest because of its biomedical and synthetic applications. In this study, we developed photoligaton of DNA by using a photoremovable protecting group and thiol-disulfide exchange reaction. A phosphoroamidite of o-nitrobenzyl derivatives were synthesized, and DNA modified with a nitrobenzyl-protected thiol group and disulfide group was synthesized by conventional phosphoroamidite chemistry using a DNA synthesizer. It was shown that photochemical reaction of a nitrobenzyl group with UV irradiation produced a free thiol group, leading to the DNA ligation through the thiol-disulfide exchange reaction.