Oral priming with oligodeoxynucleotide particles from Lactobacillus rhamnosus GG attenuates symptoms of dextran sodium sulfate-induced acute colitis in mice.

Here, we investigated the effect of prophylactic oral treatment with carbonate apatite-based particles (ID35caps) containing Lactobacillus rhamnosus GG-derived immunostimulatory oligodeoxynucleotides (ID35) when used in mice with acute colitis. Mice were administered orally with control particles (carbonate apatite particles, Caps), ID35, or ID35caps for 2 days, and then were given free access to drinking water containing 3% (w/v) dextran sodium sulfate (DSS) for 5 days (Days 0-5) to induce acute colitis. Body weight change, fecal bleeding, and stool consistency were monitored and scored as a disease activity index (DAI) to assess symptoms of colitis. On Day 10, animals were euthanized and the colon length was measured to evaluate inflammatory tissue injury. Prophylactic oral treatment with ID35caps significantly suppressed DSS-induced elevation of the DAI score and shortening of the colon compared to the respective parameters in DSS-exposed mice treated with Cap or ID35. We conclude that oral priming with ID35caps attenuates symptoms and inflammatory colonic injury in a mouse model of DSS-induced acute colitis. This finding suggests that ID35caps may be a new oral agent for preventing intestinal inflammation.

Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis.

This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides, and α-chloroamides-which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies-can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups. © 2020 Wiley Periodicals LLC. Basic Protocol: Synthesis, deprotection, cleavage, and purification of sensitive oligodeoxynucleotides Support Protocol 1: Synthesis of Dim-Dmoc nucleoside phosphoramidites Support Protocol 2: Preparation of CPG with a Dmoc linker Support Protocol 3: Synthesis of a phosphoramidite containing a sensitive alkyl ester group.

Synthesis and purification of biotinylated oligodeoxynucleotides containing single TpT dimeric pyrimidine (6-4) pyrimidone lesion.

Ultraviolet (UV) radiation could induce pyrimidine-related dimeric lesions in genomic DNA. Though the cyclobutane pyrimidine dimers (CPDs) are the most abundant UV-induced lesions, the pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) may have more serious, potentially lethal, and mutagenic effects. It is important to have 6-4PP-containing oligodeoxynucleotides to be prepared for studying their adverse biological effects. Here, we developed a UV-irradiated water droplet method for the preparation of a biotinylated, 6-4PP-containing 10-mer oligodeoxynucleotide. By the use of HPLC purification and enrichment twice, the final yield is estimated to be about 8.1%. In contrast, without applying droplet technique, the direct UV irradiation against oligonucleotide-containing aqueous solution, the product yield is very low. The enzymatic hydrolyzation of the obtained product shows a 6-4PP characteristic ion transition of 545.12 → 432.13 in negative ion mode UHPLC-Q-TOF/MS. The established procedure for the preparation of 6-4PP-containing oligonucleotides is convenient with an improved yield. Graphical abstract ᅟ.

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%.

Solid-Phase Synthesis of Oligodeoxynucleotide Analogs Containing Phosphorodithioate Linkages.

The oligodeoxynucleotide phosphorodithioate modification (PS2-ODN) uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphordiester backbone linkage. Like a natural phosphodiester ODN backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-ODNs are highly stable to nucleases and numerous in vitro assays have demonstrated their biological activity. For example, PS2-ODNs activated RNase H in vitro, strongly inhibited human immunodeficiency virus (HIV) reverse transcriptase, induced B-cell proliferation and differentiation, and bound to protein targets in the form of PS2-aptamers (thioaptamers). Thus, the interest in and promise of PS2-ODNs has spawned a variety of strategies for synthesizing, isolating, and characterizing this compounds. ODN-thiophosphoramidite monomers are commercially available from either AM Biotechnologies or Glen Research and this unit describes an effective methodology for solid-phase synthesis, deprotection, and purification of ODNs having PS2 internucleotide linkages. © 2016 by John Wiley & Sons, Inc.

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%.

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.

Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.

BACKGROUND: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

Purification of DNA-origami nanostructures by rate-zonal centrifugation.

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

[Synthesis and properties of methylene carboxamide mimetics of nucleic acids based on morpholine nucleosides].

Uracyl and adenine containing oligocarboxamide mimetics of nucleic acids based on morpholine nucleosides (MorGly) are synthesized using peptide chemistry methods. Conditions for an analysis of homogeneity of protonated at physiological pH oligomers using a capillary electrophoresis are proposed. Studies of thermostability of complementary complexes formed by MorGly oligomers revealed that melting temperature dramatically depends on heterocyclic base composition (uracyl or adenine). Cooperative interactions realized at junctions in tandem complexes give more contribution to the thermostability in the case of complexes formed by modified oligomers than native oligodeoxyriboadenilates. Adenine containing MorGly oligomers form more stable complexes with poly(U) than native oligodeoxyriboadenilate of the same length. Complexes formed by modified oligomers with polyribonucleotides are more stable in compare with polydeoxyribonucleotide.

Capillary electrophoretic separation-based approach to determine the labeling kinetics of oligodeoxynucleotides.

With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined CE protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2'-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence, the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in ssDNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.

Fast DNA interstrand cross-linking reaction by 6-vinylpurine.

Oligonucleotides that incorporate a reactive moiety to form an interstrand cross-link have been widely studied for their potential toward inhibiting gene expression or as basic tools for chemical biology studies. The 6-vinylpurine (2) newly designed in the current study serves well as a new purine-base moiety for increasing cross-link reactivity to target cytosine. Thus, oligonucleotides containing 6-vinylpurine exhibit a more selective and much smoother DNA cross-linking ability to cytosine than the oligonucleotide analogs derived from 2-amino-6-vinylpurine (1) previously explored.

In vitro base excision repair using mammalian cell extracts.

Base excision repair (BER) is an essential cellular mechanism that maintains genome stability by repairing DNA damage, such as DNA base lesions, base loss (AP sites) and single strand breaks, generated through endogenous metabolism or via exogenous mutagens. Therefore, in vitro BER assays are important for our understanding of the mechanism of cellular response to mutagens and may also reveal important information about the development of several DNA repair-related human diseases, such as cancer, and aging. Here, we describe the preparation and use of mammalian cell extracts in in vitro BER assays using both oligonucleotide and closed circular DNA substrates containing site-specific DNA lesions, in combination with denaturing acrylamide gel electrophoresis and phosphor imaging analysis.

Purification of synthetic oligonucleotides via catching by polymerization.

This unit describes the purification of synthetic oligodeoxyribonucleotides (ODN) using a catching-by-polymerization approach. In a crude ODN, the major impurity is the failure sequences generated in the coupling step of each synthetic cycle. They are difficult to remove due to the similarity of their physical properties to the full-length sequences. Two non-chromatographic methods are described in the unit to solve the problem. In the first one, during automated synthesis, the failure sequences are tagged with a methacrylamide group, which is polymerizable and can participate in acrylamide radical polymerization reactions; the full-length sequences are not tagged. After synthesis, the crude mixture is subjected to polymerization. The failure sequences are incorporated into an insoluble polymer; the full-length sequences are extracted with water. In the second method, the full-length sequences are tagged with a methacrylamide group via a cleavable linker; the failure sequences are not tagged. After synthesis, the full-length sequences are incorporated into a polymer; the failure sequences are washed away with water. Pure full-length sequences are cleaved from the polymer. The two methods are complementary. .

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp.

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

In vitro selection of metal ion-selective DNAzymes.

The discovery of DNAzymes that can catalyze a wide range of reactions in the presence of metal ions is important on both fundamental and practical levels; it advances our understanding of metal-nucleic acid interactions and allows for the design of highly sensitive and selective metal ion sensors. A crucial factor in this success is a technique known as in vitro selection, which can rapidly select metal-specific RNA-cleaving DNAzymes. In vitro selection is an iterative process where a DNA pool containing a random region is incubated with the target metal ion. Those DNA sequences that catalyze the preferred reaction (the "winners") are amplified and carried on to the next step, where the selection is carried out under more stringent conditions. In this way, the selection pool becomes enriched with DNAzymes that exhibit desirable activity and selectivity. The method described can be applied to isolate DNAzymes selective to many different types of metal ions or different oxidation states of the same metal ion.

TopDown real-time gene synthesis.

This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.

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.

Synthesis, purification, and characterization of immune-modulatory oligodeoxynucleotides that act as agonists of Toll-like receptor 9.

Methods and protocols for automated synthesis and purification of immune modulatory oligonucleotides (IMOs), a novel class of Toll-like receptor 9 (TLR9) agonists, are described. IMOs containing two short identical sequences of 11-mers with phosphorothioate linkages can be synthesized in parallel synthetic strategy. A C3-linker that mimics the natural inter-nucleotide distance was commonly used for joining the two segments of IMOs. NittoPhase solid support bearing a symmetrical C3-linker (glycerol) and nucleoside-ß-cyanoethyl-N,N-diisopropylphosphoramidites were used for IMO synthesis. The parallel synthesis was carried out in a 3'→ 5' direction with removal of the final dimethoxytrityl (DMT) protecting group. After synthesis, the IMO was cleaved and deprotected by treating with aqueous ammonia. The product was purified on anion-exchange HPLC, desalted, lyophilized, and characterized by anion-exchange HPLC, capillary gel electrophoresis, polyacrylamide gel electrophoresis, and MALDI-TOF mass spectral analysis.

[Modulation of Toll-like signal path of allergic asthma by CpG-ODNs from Bordetella pertussis].

This study focused on prevention and treatment of acute and chronic asthma by oligonucleotides containing unmethylated CpG motifs (CpG-ODNs). Acute and chronic asthma models of mice were made by sensitizing and inhaling ovalbumin (OVA); The number of white blood cells (WBC) and eosnophils (EOS) in bronchoalveolar lavage fluid (BALF) was counted and the concentration of cytokines and vascular endothelial growth factor (VEGF) was examined in BALF by ELISA kit. After that, TLR-9 mRNA was detected in mice spleen cells by reverse transcription polymerase chain reaction (RT-PCR) and TLR-9 protein was determined in mice lung tissues by Western blotting. Compared with acute asthma models of mice, WBC in BALF decreased obviously in the groups of Bordetella pertussis, CpG-ODNs and seq A to seq I which were administrated by both of intragastric (ig) and intraperitoneal (ip) injection group, EOS decreased obviously in Bordetella pertussis, CpG+ and seq A to seq D ig groups, and in all ip administrating groups, although it was not effective in the groups of seq E to seq I. In chronic asthma models of mice, IFN-gamma increased ((1) control: 176.45 +/- 23.46 pg x mL(-1); (2) model: 174.11 +/- 22.71 pg x mL(-1); (3) CpG+ ip: 220.56 +/- 15.42 pg x mL(-1); (4) seq A ip: 225.23 +/- 21.60 pg x mL(-1)) and IL-4 decreased obviously (1) control: 66.91 +/- 5.81 pg x mL(-1); (2) model: 81.02 +/- 11.24 pg x mL(-1); (3) CpG+ ip: 63.99 +/- 6.09 pg x mL(-1); (4) seq A ip: 62.75 +/- 10.03 pg x mL(-1)) in the BALF of CpG+ and seq A ip group, although VEGF was not changed in this research. And also, TLR-9 mRNA in spleen cells (TLR-9/GAPDH: (1) control: 0.62 +/- 0.13; (2) model: 0.66 +/- 0.17; (3) CpG+ ip: 1.46 +/- 0.26; (4) seq A ip: 1.42 +/- 0.34) and TLR-9 protein in lung tissues (TLR-9/beta-actin: (1) control: 0.63 +/- 0.16; (2) model: 0.61 +/- 0.07; (3) CpG+ ip: 1.15 +/- 0.25; (4) seq A ip: 1.03 +/- 0.29) both increased in ip groups, but the change was not significant in ig group. The study confirms that CpG-ODNs and seq A could inhibit airway inflammation remarkably, this mechanism might be related with regulating Th1/Th2 balance and controlling the expression of TLR-9.