A new approach for the improvement of separation of oligonucleotides by recycling ion-pairing chromatography is described. In the so-called repetto process, segments of separated compounds are sequentially returned to the inlet for multiple passages through the column without a need to pass a pump and with the possibility of detecting the level of separation between individual passages. Unlike in the recently described twin-column recycle approach in which eluents are repeatedly transferred between two separation columns, with the repetto method a single column is sufficient, and the detector is not exposed to high back pressure. The repetto principle was used for the separation of synthetic oligonucleotides, resulting in a multi-fold improvement in single nt resolution of long (> 50 nt) synthetic oligonucleotide fragments with high gas chromatography (guanine-cytosine) content > 40% and their separation from impurities of the original synthesis.
- Klíčová slova
- HPLC, TEAA, ion‐pairing chromatography, oligonucleotides, recycling, repetto,
- MeSH
- oligonukleotidy * izolace a purifikace analýza chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- oligonukleotidy * MeSH
Fast and reasonable low-scale (200 nmol) syringe-made synthesis of 15 N-labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid-phase controlled pore glass synthesis column technique via the phosphoramidite method. Twenty-four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.
- Klíčová slova
- 15N-labeled oligonucleotides, low-scale, phosphoramidite synthesis, syringe-made codon synthesis,
- MeSH
- DNA chemie MeSH
- hmotnostní spektrometrie MeSH
- injekční stříkačky * MeSH
- kodon MeSH
- oligonukleotidy * chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- kodon MeSH
- oligonukleotidy * MeSH
Ionic liquids (ILs) have become nearly ubiquitous solvents and their interactions with biomolecules has been a focus of study. Here, we used the fluorescence emission of DAPI, a groove binding fluorophore, coupled with molecular dynamics (MD) simulations to report on interactions between imidazolium chloride ([Imn,1]+) ionic liquids and a synthetic DNA oligonucleotide composed entirely of T/A bases (7(TA)) to elucidate the effects ILs on a model DNA duplex. Spectral shifts on the order of 500-1000 cm-1, spectral broadening (~1000 cm-1), and excitation and emission intensity ratio changes combine to give evidence of an increased DAPI environment heterogeneity on added IL. Fluorescence lifetimes for DAPI/IL solutions yielded two time constants 0.15 ns (~80% to 60% contribution) and 2.36-2.71 ns for IL up to 250 mM. With DNA, three time constants were required that varied with added IL (0.33-0.15 ns (1-58% contribution), ~1.7-1.0 ns (~5% contribution), and 3.8-3.6 ns (94-39% contribution)). MD radial distribution functions revealed that π-π stacking interactions between the imidazolium ring were dominant at lower IL concentration and that electrostatic and hydrophobic interactions become more prominent as IL concentration increased. Alkyl chain alignment with DNA and IL-IL interactions also varied with IL. Collectively, our data showed that, at low IL concentration, IL was primarily bound to the DNA minor groove and with increased IL concentration the phosphate regions and major groove binding sites were also important contributors to the complete set of IL-DNA duplex interactions.
- Klíčová slova
- DAPI, DNA duplex, DNA oligonucleotide, fluorescence, fluorescence lifetime, imidazolium ionic liquids, molecular dynamics, radial distribution function,
- MeSH
- DNA chemie MeSH
- imidazoly chemie MeSH
- iontové kapaliny chemie MeSH
- oligonukleotidy chemie MeSH
- simulace molekulární dynamiky * MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- imidazole MeSH Prohlížeč
- imidazoly MeSH
- iontové kapaliny MeSH
- oligonukleotidy MeSH
Non-canonical forms of nucleic acids represent challenging objects for both structure-determination and investigation of their potential role in living systems. In this work, we uncover a structure adopted by GA repetition locked in a parallel homoduplex by an i-motif. A series of DNA oligonucleotides comprising GAGA segment and C3 clip is analyzed by NMR and CD spectroscopies to understand the sequence-structure-stability relationships. We demonstrate how the relative position of the homopurine GAGA segment and the C3 clip as well as single-base mutations (guanine deamination and cytosine methylation) affect base pairing arrangement of purines, i-motif topology and overall stability. We focus on oligonucleotides C3GAGA and methylated GAGAC3 exhibiting the highest stability and structural uniformity which allowed determination of high-resolution structures further analyzed by unbiased molecular dynamics simulation. We describe sequence-specific supramolecular interactions on the junction between homoduplex and i-motif blocks that contribute to the overall stability of the structures. The results show that the distinct structural motifs can not only coexist in the tight neighborhood within the same molecule but even mutually support their formation. Our findings are expected to have general validity and could serve as guides in future structure and stability investigations of nucleic acids.
The Gram-negative, obligate intracellular tick-transmitted pathogen Anaplasma phagocytophilum can cause acute febrile diseases in humans and domestic animals. The expansion of the tick Ixodes ricinus (Linnaeus, 1758) in northern Europe due to climate change is of serious concern for animal and human health. The aim of the present study was to investigate the impact of A. phagocytophilum infection in moose Alces alces (Linnaeus) calves by evaluating the carcass weights of infected and non-infected animals and examining animal tissues samples for co-infections with either species of Babesia Starcovici, 1893 or bacteria of the genus Bartonella. The carcasses of 68 free-ranging moose calves were weighed by hunters during the hunting seasons from 2014 to 2017 in two regions in southern Norway and spleen samples were collected. Anaplasma phagocytophilum was detected in moose sampled from locations infected with ticks with a prevalence of 82% (n = 46). The carcass weights of A. phagocytophilum-infected calves (n = 46) and non-infected (n = 22) calves were compared. Although the average weight of infected calves (45.6 kg) was lower than that of non-infected calves (46.5 kg), the difference was not statistically significant. Three different variants of the bacterium 16S rRNA gene were identified. The average weight of animals infected with variant I was 49.9 kg, whereas that of animals infected with variant III was 42.0 kg, but the difference was not statistically significant (p = 0.077). Co-infections of A. phagocytophilum with Bartonella spp. or with Babesia spp. were found in 20 and two calves, respectively. A triple infection was found in two calves. Sequence analysis of the 18S rRNA gene of Babesia-positive samples revealed the presence of Babesia cf. odocoilei (Emerson et Wright, 1970). Strains of Bartonella closely related to Bartonella bovis (Bermond, Boulouis, Heller, Laere, Monteil, Chomel, Sander, Dehio et Piemont, 2002) were identified based on phylogenetic analysis of the gltA and rpoB genes. The loss of body mass in moose calves in the tick-infected site was probably influenced by multiple factors.
- Klíčová slova
- Babesia spp., Bartonella spp., body condition, calf weight, co-infections,
- MeSH
- Anaplasma phagocytophilum * klasifikace genetika izolace a purifikace MeSH
- Babesia genetika MeSH
- Bartonella genetika MeSH
- DNA bakterií chemie genetika izolace a purifikace MeSH
- ehrlichióza komplikace epidemiologie patologie veterinární MeSH
- fylogeneze MeSH
- oligonukleotidy chemie MeSH
- polymerázová řetězová reakce veterinární MeSH
- sekvence nukleotidů MeSH
- slezina mikrobiologie patologie MeSH
- tělesná hmotnost MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko epidemiologie MeSH
- Názvy látek
- DNA bakterií MeSH
- oligonukleotidy MeSH
Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.
- MeSH
- cirkulární dichroismus MeSH
- DNA genetika ultrastruktura MeSH
- G-kvadruplexy * MeSH
- konformace nukleové kyseliny MeSH
- nukleotidy chemie genetika MeSH
- oligonukleotidy chemie genetika MeSH
- polymorfismus genetický genetika MeSH
- RNA genetika ultrastruktura MeSH
- simulace molekulární dynamiky MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- nukleotidy MeSH
- oligonukleotidy MeSH
- RNA MeSH
5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2'-deoxycytidine and uridine 3'-O-phosphoramidites and their use in automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as primers for PCR to construct DNA templates modified in the promoter region that allowed switching of transcription through photochemical uncaging.
- MeSH
- 5-methylcytosin analogy a deriváty chemická syntéza chemie MeSH
- deoxycytidin chemie MeSH
- DNA chemie MeSH
- epigeneze genetická MeSH
- epigenomika MeSH
- molekulární struktura MeSH
- oligonukleotidy chemická syntéza chemie MeSH
- organofosforové sloučeniny chemie MeSH
- pyrimidinové nukleosidy chemie MeSH
- uracil chemie MeSH
- uridin analogy a deriváty chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 5-hydroxymethylcytosine MeSH Prohlížeč
- 5-methylcytosin MeSH
- deoxycytidin MeSH
- DNA MeSH
- oligonukleotidy MeSH
- organofosforové sloučeniny MeSH
- phosphoramidite MeSH Prohlížeč
- pyrimidinové nukleosidy MeSH
- uracil MeSH
- uridin MeSH
- uridine phosphoramidite MeSH Prohlížeč
A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN-TFO hybrids is based on application of the alkyne-azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5'- or 3'-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN-TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34 %) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN-TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity.
- Klíčová slova
- DNA cleavage, click chemistry, copper, nucleases, oligonucleotides,
- MeSH
- deoxyribonukleasy chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- fenantroliny chemická syntéza chemie MeSH
- konformace nukleové kyseliny MeSH
- oligonukleotidy chemie MeSH
- sekvence nukleotidů MeSH
- štěpení DNA * MeSH
- syntetická chemie okamžité shody MeSH
- tranzitní teplota MeSH
- ultrafialové záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- deoxyribonukleasy MeSH
- DNA MeSH
- fenantroliny MeSH
- oligonukleotidy MeSH
- triplex DNA MeSH Prohlížeč
Taking advantage of surface-enhanced Raman scattering (SERS) methodology with its unique ability to collect abundant intrinsic fingerprint information and noninvasive data acquisition we set up a SERS-based approach for recognition of physically induced DNA damage with further incorporation of artificial neural network (ANN). As a proof-of-concept application, we used the DNA molecules, where the one oligonucleotide (OND) was grafted to the plasmonic surface while complimentary OND was exposed to UV illumination with various exposure doses and further hybridized with the grafted counterpart. All SERS spectra of entrapped DNA were collected by several operators using the portable spectrometer, without any optimization of measurements procedure (e.g., optimization of acquisition time, laser intensity, finding of optimal place on substrate, manual baseline correction, etc.) which usually takes a significant amount of operator's time. The SERS spectra were employed as input data for ANN training, and the performance of the system was verified by predicting the class labels for SERS validation data, using a spectra dataset, which has not been involved in the training process. During that phase, accuracy higher than 98% was achieved with a level of confidence exceeding 95%. It should be noted that utilization of the proposed functional-SERS/ANN approach allows identifying even the minor DNA damage, almost invisible by control measurements, performed with common analytical procedures. Moreover, we introduce the advanced ANN design, which allows not only classifying the samples but also providing the ANN analysis feedback, which associates the spectral changes and chemical transformations of DNA structure.
- Klíčová slova
- Artificial neural network, DNA, Detection and recognition, Photo-damage, SERS,
- MeSH
- biosenzitivní techniky * MeSH
- DNA chemie izolace a purifikace MeSH
- kovové nanočástice chemie MeSH
- neuronové sítě (počítačové) MeSH
- oligonukleotidy chemie MeSH
- poškození DNA * MeSH
- Ramanova spektroskopie * MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- oligonukleotidy MeSH
- zlato MeSH
We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.
- Klíčová slova
- H-phosphinate, H-phosphonate, oligonucleotide, phosphonamidate, phosphonate, phosphonothioate, phosphoramidate, phosphoramidite, phosphorothioate,
- MeSH
- amidy chemie MeSH
- dimerizace MeSH
- fosfáty chemie MeSH
- fosforothioátové oligonukleotidy chemická syntéza MeSH
- kyseliny fosforečné chemie MeSH
- molekulární struktura MeSH
- oligonukleotidy chemická syntéza chemie MeSH
- techniky syntézy na pevné fázi * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- amidy MeSH
- fosfáty MeSH
- fosforothioátové oligonukleotidy MeSH
- kyseliny fosforečné MeSH
- oligonukleotidy MeSH
- phosphoramidic acid MeSH Prohlížeč
- phosphorodithioic acid MeSH Prohlížeč
