基于化学衍生-质谱技术的生物与临床样本中核酸修饰分析

除了经典碱基外,核酸(DNA和RNA)中还包含许多化学修饰。迄今为止,已经在核酸中鉴定了超过150多种化学修饰。这些化学修饰不会改变核酸的序列,但会改变它们的结构和生化特性,最终调节基因的时空表达。阐明这些修饰的功能可以促进对生命体生理调控机制的深入认识和理解。然而,核酸修饰在体内的丰度通常很低。因此,高灵敏和特异的检测方法对破译这些修饰的功能至关重要。化学衍生与质谱技术相结合对内源性低丰度核酸修饰展现出很好的分析能力。在过去几年中,研究者建立了多种基于化学衍生-质谱分析的分析方法,用于灵敏、高效地分析核酸修饰。该文总结了通过化学衍生-质谱分析方法来破译核酸修饰的最新进展,希望能促进未来对核酸修饰功能的深入研究。     

Deciphering nucleic acid modifications by chemical derivatization-mass spectrometry analysis

Chemical derivatization in combination with mass spectrometry (MS) analysis is a promising strategy for the sensitive and effective analysis of nucleic acid modifications. In this review, we summarize the recent advances for deciphering modifications in DNA and RNA by chemical derivatization-MS analysis.     

Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds-III

Six polymorphic compounds, having between two and five modifications, are described. Enantiotropic modifications were found for three of them: 9-fluorenylmethanol (2 modifications), polyester red A (3 modifications) and polyester red B (3 modifications). There were no indications of enantiotropy in the other three: diphenylcarbamoyl chloride (4 modifications), 4-hydroxyphenylacetic acid (5 modifications) and 2-hydroxy-1-naphthalenecarbaldehyde (5 modifications).     

Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds—VI

Four of the six polymorphic compounds described occur in an enantiotropic low temperature form in the commercial products. Only for two of them is the highest melting form the stable form at room temperature. Enantiotropy between the commercial form and the higher melting modifications was observed for aminoacetonitrile hydrochloride (3 modifications), 3,4-dinitrobenzoic acid (4 modifications), 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine (3 modifications) and (2-furyl)oxoacetamide (3 modifications). 2-Biphenyl-5-phenyl-1,3,4-oxadiazole (5 modifications) and (+-)-4-chloromandelic acid (3 modifications) can be assumed to be monotropic, but exact information about the modifications was impossible to obtain.     

Analytical methods for locating modifications in nucleic acids

We reviewed and summarized the established methods and the breakthrough of the techniques for locating modifications in nucleic acids. In addition, we discussed the principles, applications, advantages and drawbacks of these methods.     

Comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry

RNA molecules contain diverse modifications that display important functions in a variety of physiological and pathological processes. So far over 150 chemical modifications have been characterized to be present in various RNA species, such as in messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). Previous studies revealed that certain RNA modifications were correlated to specific human diseases, indicating RNA modifications could serve as the potential indicator of human diseases. However, systemic investigation of the alteration of RNA modifications in different RNA species of carcinoma tissues are still lacked. Herein, we carried out the comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The developed method allowed us to simultaneously detect 48 different types of RNA modifications. Using this method, we detected 10, 15, 14, and 25 modifications in mRNA, 18S rRNA, 28S rRNA and small RNA (< 200 nt), respectively. Compared to the normal tissues, we revealed a total of 14 RNA modification exhibited significant increase and 2 RNA modifications showed significant decrease in thyroid carcinoma tissues. Our study provided the first comprehensive profile as well as the alteration of modifications in different RNA species in thyroid carcinoma and matched tumor-adjacent normal tissues. The altered pattern RNA modifications may serve as the indicator of thyroid carcinoma. Moreover, this study may promote the in-depth understanding of the regulatory roles of RNA modifications in thyroid carcinoma.     

Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds—VIII

Five polymorphic organic compounds which exhibited from three to seven modifications were examined. Because of enantiotropy, the commercial forms of three of the compounds did not consist of the modification with the highest melting point: These were 4-hydroxy-3,5-dimethoxybenzoic acid (4 modifications), 3-pyridylacetic acid (4 modifications) and 2,4,5-triaminopyrimidine (4 modifications). In contrast, 2-amino-2-cyanoacetamide (3 modifications) and [([(4-methylphenyl)sulphonyl]oxy)imino]propanedinitrile (7 modifications) were present in the highest melting form in the commercial products. A further 32 compounds (most of which had only two modifications) were examined and are reported without detailed results; six of them were enantiotropic.     

Adolescent alcohol exposure alters DNA and RNA modifications in peripheral blood by liquid chromatography-tandem mass spectrometry analysis

Alcohol consumption is a critical risk factor contributing to a verity of human diseases. The incidence of alcohol use disorder increases across adolescence in recent years. Accumulating line of evidence suggests that alcohol-induced changes of DNA cytosine methylation(5-methyl-2-deoxycytidine, 5 m C) in genomes play an important role in the development of diseases. However, systemic investigation of the effects of adolescent alcohol exposure on DNA and RNA modifications is still lacked. Especia...     

Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end.     

Profiling of Ribose Methylations in RNA by High‐Throughput Sequencing

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.     

Protein posttranslational modifications: the chemistry of proteome diversifications

The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.     

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully defined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. Notably, among these identified modifications, 2 kinds of modifications of N²,N²-dimethylguanosine (m^2,2G) and N⁶,N⁶-dimethyladenosine (m^6,6A) in 18S rRNA, and 4 kinds of modifications of m^2,2G, m^6,6A, N7-methylguanosine (m⁷G) and 3-methyluridin (m³U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.     

Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds—V

All the polymorphic compounds described are enantiotropic, so that in four cases, order-disorder or rotational transformation can be deduced on the basis of the relationships between enthalpy of transformation and enthalpy of fusion. The following compounds are of this type: (±)-1-azabicyclo- [2.2.2]-octan-3-ol (4 modifications), 5-norbornene-2,3-dicarboxylic anhydride (3 modifications), (l.R)-(–)-camphorquinone (3 modifications) and 2-bromoethylamine hydrobromide (3 modifications). For tris(acetylacetonato)aluminium(III) (3 modifications) and 2-(4-aminophenyl)-6-methylbenzothiazole (5 modifications), another type of transformation must be assumed.     

Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds—VII

For four of the polymorphic compounds described, the commercial product does not consist of the form with the highest melting point. A spontaneous enantiotropic transformation is observed for 2-benzoxazolethiol (three modifications). For 2-furancarboxylic acid (three modifications) and 4-methoxy-3-nitrobenzaldehyde (three modifications), the form with the lower melting point either melts inhomogeneously with crystallization of modificationI, or homogeneously without formation of the highest melting form. Monotropy is observed for 2-nitrophenylacetic acid (four modifications) and salicylic acid hydrazide (four modifications). A special case is 1,2,4-triazole-3-thiol (three modifications) which in the commercial product occurs mainly as the metastable modificationIII, which is monotropic, whereas there is enantiotropy between modificationsI andII.     

Conformational and packing energy for the three crystalline forms of isotactic poly-α-butene

Calculations of the conformational and of the packing energy have been performed for the three main crystalline modifications of isotactic poly-α-butene. Calculations of the conformational energy have been performed first through minimizations of the energy of the isolated chains under the constraint of maintaining the experimental helical symmetry and periodicity for each modification. In this way accurate conformations have been determined for the helices corresponding to the three modifications. These conformations have been used for calculation of the packing energy of the three modifications of poly-α-butene. The considered space groups are those experimentally observed; for each modification also the possibility of statistical up and down positioning of the chains has been examined. The differences between the packing energies are greater than those between the conformational energies for the three different modifications. The relative stabilities of the three modifications of poly-α-butene, as obtained by our calculations, are discussed.     

Designed post-self-assembly structural and functional modifications of a truncated tetrahedron

Post-self-assembly modifications of a discrete metal-organic supramolecular structure have been developed. Such modifications allow the properties of the self-assembled supramolecular species to be changed in a simple and efficient manner (>90% yield). Initiated by the application of chemical stimuli, the post-self-assembly modifications described herein result in three distinct changes to the supramolecular system: an individual building-block component change, an overall structural modification, and a functional evolution of a [6+4] metal-organic supramolecular structure. The three modifications have been carefully examined by a range of characterization methods, including NMR and UV-vis spectroscopy, electrospray ionization mass spectrometry, pulsed field gradient spin echo NMR measurements, electrochemical analysis, and computational simulations.