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1.
J Am Chem Soc ; 139(5): 1766-1769, 2017 02 08.
Article in English | MEDLINE | ID: mdl-28107630

ABSTRACT

5-Hydroxymethylcytidine (hm5C) was recently identified as a direct metabolite of m5C in RNA. We investigated the stability of hm5C in human cells using bio-isotopologues and LC-MS/HRMS. This has led to the discovery of a second oxidative metabolite of m5C in RNA, namely 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm). Subsequent quantitative analysis of total RNA from higher organisms revealed varying levels and TET-independent formation of this new RNA modification.


Subject(s)
Cytidine/analogs & derivatives , RNA/chemistry , Animals , Brain/metabolism , Chromatography, Liquid , Cytidine/chemistry , Cytidine/metabolism , HEK293 Cells , Humans , Mice , Molecular Structure , Oxidation-Reduction , RNA/metabolism , Tandem Mass Spectrometry
2.
Chembiochem ; 18(11): 979-984, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28449301

ABSTRACT

More than a hundred distinct modified nucleosides have been identified in RNA, but little is known about their distribution across different organisms, their dynamic nature and their response to cellular and environmental stress. Mass-spectrometry-based methods have been at the forefront of identifying and quantifying modified nucleosides. However, they often require synthetic reference standards, which do not exist in the case of many modified nucleosides, and this therefore impedes their analysis. Here we use a metabolic labelling approach to achieve rapid generation of bio-isotopologues of the complete Caenorhabditis elegans transcriptome and its modifications and use them as reference standards to characterise the RNA modification profile in this multicellular organism through an untargeted liquid-chromatography tandem high-resolution mass spectrometry (LC-HRMS) approach. We furthermore show that several of these RNA modifications have a dynamic response to environmental stress and that, in particular, changes in the tRNA wobble base modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5 s2 U) lead to codon-biased gene-expression changes in starved animals.


Subject(s)
RNA Processing, Post-Transcriptional , Stress, Physiological/genetics , Transcriptome , Animals , Caenorhabditis elegans , Chromatography, Liquid , Isotope Labeling , Tandem Mass Spectrometry , Thiouridine/analogs & derivatives , Thiouridine/metabolism
3.
Angew Chem Int Ed Engl ; 55(37): 11144-8, 2016 09 05.
Article in English | MEDLINE | ID: mdl-27440712

ABSTRACT

Biopolymers are an attractive alternative to store and circulate information. DNA, for example, combines remarkable longevity with high data storage densities and has been demonstrated as a means for preserving digital information. Inspired by the dynamic, biological regulation of (epi)genetic information, we herein present how binary data can undergo controlled changes when encoded in synthetic DNA strands. By exploiting differential kinetics of hydrolytic deamination reactions of cytosine and its naturally occurring derivatives, we demonstrate how multiple layers of information can be stored in a single DNA template. Moreover, we show that controlled redox reactions allow for interconversion of these DNA-encoded layers of information. Overall, such interlacing of multiple messages on synthetic DNA libraries showcases the potential of chemical reactions to manipulate digital information on (bio)polymers.


Subject(s)
DNA/genetics , Epigenomics , Information Storage and Retrieval/methods , Biopolymers/chemistry , DNA/chemistry
4.
Chembiochem ; 16(5): 752-5, 2015 Mar 23.
Article in English | MEDLINE | ID: mdl-25676849

ABSTRACT

RNA methylation is emerging as a regulatory RNA modification that could have important roles in the control and coordination of gene transcription and protein translation. Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m(5)C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm(5)C) and 5-formylcytidine (f(5)C). Furthermore, we have identified hm(5)C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells. This suggests m(5)C oxidation is a conserved process that could have critical regulatory functions inside cells.


Subject(s)
Cytosine/analogs & derivatives , RNA/chemistry , RNA/metabolism , 5-Methylcytosine/analogs & derivatives , Animals , Chromatography, High Pressure Liquid , Cytosine/biosynthesis , Cytosine/chemistry , Cytosine/metabolism , Mice , Mice, Inbred C57BL , Molecular Structure , Oxidation-Reduction , Tandem Mass Spectrometry
5.
J Org Chem ; 80(9): 4553-65, 2015 May 01.
Article in English | MEDLINE | ID: mdl-25826382

ABSTRACT

The Lewis acid mediated reduction of ribose-, arabinose-, xylose-, and lyxose-derived methyl and phenyl ketofuranoses with triethylsilane as nucleophile was found to proceed with good to excellent stereoselectivity to provide the 1,2-cis addition products. The methyl ketoses reacted in a more stereoselective manner than their phenyl counterparts. The stereochemical outcome of the reactions parallels the relative stability of the oxocarbenium ion conformers involved, as assessed by calculating the free energy surface maps of their complete conformational space. The Lewis acid mediated reduction allows for a direct synthesis of C-glycosides with predictable stereochemistry.

7.
Angew Chem Int Ed Engl ; 53(39): 10381-5, 2014 Sep 22.
Article in English | MEDLINE | ID: mdl-25081839

ABSTRACT

Lewis acid mediated substitution reactions using [D]triethylsilane as a nucleophile at the anomeric center of the four pentofuranoses, ribose, arabinose, xylose, and lyxose, all proceed with good to excellent stereoselectivity to provide the 1,2-cis adducts. To unravel the stereoelectronic effects underlying the striking stereoselectivity in these reactions we have mapped the energy landscapes of the complete conformational space of the oxocarbenium ions of the four pentofuranoses. The potential energy surface maps provide a detailed picture of the influence of the differently oriented substituents and their mutual interactions on the stability of the oxocarbenium ions and the maps can be used to account for the observed stereoselectivities of the addition reactions.

8.
J Chromatogr A ; 1730: 465137, 2024 Aug 16.
Article in English | MEDLINE | ID: mdl-38996514

ABSTRACT

End groups of poly(Lactide-co-glycolide) (PLGA) play an important role in determining the properties of polymers for use in drug delivery systems. For instance, it has been reported that the encapsulation efficiency in PLGA microspheres varies significantly between ester-terminated and acid-terminated PLGA. More importantly, the in-vivo degradation time of such polymer excipients is influenced by the functional end-group of the copolymer used. The end group distribution in PLGA polymers has been studied using electrospray and matrix-assisted laser-desorption/ionization - high-resolution mass spectrometry. In both cases, the application of these methods is typically limited to PLGA having a molecular weight of up to 4 kDa. 13Carbon-nuclear-magnetic-resonance has also been reported as a method to differentiate and quantify PLGA end groups with a molecular weight up to 136 kDa. However, reported NMR methods take over 12 h per sample, limiting throughput.Cryoprobe NMR can reduce the time required for the process, however such NMR equipment is costly, which makes it unsuitable for the quality control of PLGA. Here, we present a normal-phase liquid chromatography method capable of resolving functionality type distribution (FTD) and, partially, chemical composition distribution (CCD) in commercial PLGA polymers obtained from ring opening polymerization. This method can separate PLGA polymers with a molecular weight of up to 183.0 kDa while also enabling the simultaneous separation of the difference of Lactic acid (LA)/Glycolic acid (GA) ratios. To achieve this, a cross-linked diol column was used with a ternary gradient from HEX to 0.1 % v/v TEA in EA to 0.1 % v/v FA in THF to allow first for the elution of mono-ester terminated PLGA, followed by the di-acid terminated. In addition, a separation of ester-terminated PLGA in the difference of the LA/GA ratio was achieved. This method is expected to aid in understanding the correlation between PLGA's FTD, CCD, and physical properties, facilitating product development and quality control.


Subject(s)
Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Molecular Weight , Lactic Acid/chemistry , Chromatography, Liquid/methods , Magnetic Resonance Spectroscopy , Hydrogen-Ion Concentration
9.
Nat Chem ; 11(7): 629-637, 2019 07.
Article in English | MEDLINE | ID: mdl-31209299

ABSTRACT

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.


Subject(s)
DNA/genetics , Genome/genetics , Sequence Analysis, DNA/methods , Aldehydes/chemistry , Base Sequence , DNA/chemistry , DNA Damage/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Leishmania major/genetics , Molecular Probes/chemical synthesis , Molecular Probes/chemistry , Thymine/chemistry , Uracil-DNA Glycosidase/chemistry
10.
J Org Chem ; 73(23): 9458-60, 2008 Dec 05.
Article in English | MEDLINE | ID: mdl-18991380

ABSTRACT

A new method for the construction of pyrophosphates is reported based on the coupling of a sugar phosphate and a nucleoside phosphoramidite. The in situ formed phosphate-phosphite intermediate was subsequently oxidized with tBuOOH. Three UDP-N-acetylglucosamine derivatives were prepared using this one-pot procedure in good yields.


Subject(s)
Carbohydrates/chemistry , Organophosphorus Compounds/chemistry , Acetylglucosamine/chemistry , Chemistry, Organic/methods , Magnetic Resonance Spectroscopy/methods , Models, Chemical , Nucleosides/chemistry , Nucleotides/chemistry , Phosphates/chemistry , Phosphites/chemistry , Uridine Diphosphate/chemistry
11.
Genome Biol ; 18(1): 23, 2017 01 30.
Article in English | MEDLINE | ID: mdl-28137275

ABSTRACT

BACKGROUND: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. RESULTS: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(ß-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases. CONCLUSIONS: By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.


Subject(s)
Chromosome Mapping/methods , DNA, Protozoan/genetics , Leishmania/genetics , Pentoxyl/analogs & derivatives , DNA, Protozoan/chemistry , Glucosides/chemistry , Leishmania/chemistry , Pentoxyl/chemistry , Uracil/analogs & derivatives , Uracil/chemistry
12.
PLoS One ; 11(3): e0152322, 2016.
Article in English | MEDLINE | ID: mdl-27031619

ABSTRACT

Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.


Subject(s)
DNA/metabolism , Plasmodium berghei/genetics , Sequence Analysis, DNA/methods , 5-Methylcytosine/metabolism , Chromatography, High Pressure Liquid , CpG Islands , DNA/analysis , DNA/chemistry , DNA Methylation , Gene Library , Sulfites/chemistry , Tandem Mass Spectrometry
14.
Org Lett ; 12(23): 5486-9, 2010 Dec 03.
Article in English | MEDLINE | ID: mdl-21049910

ABSTRACT

The conjugation of a ribonucleic acid 16-mer with the cationic amphiphilic peptide penetratin and an anionic hyaluronan tetrasaccharide by means of Cu-free "click" chemistry is reported. The alkyne-functionalized 16-mer was prepared by automated solid-phase synthesis, using a newly developed strained cyclooctyne phosphoramidite in the final coupling. Cycloaddition of the alkyne functionalized RNA to the azide containing biomolecules led to a clean conversion into the corresponding nucleic acid conjugates.


Subject(s)
Cyclooctanes/chemistry , Organophosphorus Compounds/chemistry , RNA/chemical synthesis , Molecular Structure
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