Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Scherer, Florian; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi Jakob; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

doi:10.1038/nchem.2307

Cited by 43 publications

(59 citation statements)

References 31 publications

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“…4MPA ( 139 ) was used successfully to enhance the removal of hemiaminal adducts and aminal cross-links from RNA in vitro and enabled the recovery of considerably greater quantities of RNA amplification signals from preserved tissue. 165 …”

Section: Catalysts For Oxime/hydrazone Formationmentioning

confidence: 99%

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

2017

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The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chemistry, biomaterials and hydrogels, dynamic combinatorial chemistry, organic synthesis, and chemical biology. Here we outline chemical developments in this field, with special focus on the past ∼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomolecules are described. The basic chemical properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equilibria in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified especially fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addition, the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A number of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomolecules and biomaterials.

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Section: Catalysts For Oxime/hydrazone Formationmentioning

confidence: 99%

Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis

2017

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“…Regarding the rate of modification of NMPs, data in the literature are heterogeneous with the limitation that most studies analyzed DNA and reported data from in vitro reaction and not from ex vivo analysis. Masuda and co-workers found that Adenine and Cytosine were the most modified nitrogenous bases after in vitro treatment of RNA with formaldehyde [ 7 ], while Karmakar and colleagues focused their study mostly on AMP and dAMP [ 24 ]. Our results on the in vitro conversion of NMPs by formaldehyde rank AMP modification (66%) above CMP (50%) and GMP (36%).…”

Section: Discussionmentioning

confidence: 99%

“…It is also possible that the dehydration process in alcohol may play a role in the conversion of formaldehyde-modified nucleic acids [ 26 ], which of course is lacking in pure formalin-fixed samples. Lastly, signals at higher retention times (>40 min) in our chromatograms are likely due to bridging irreversible dimers of amino bases [ 24 ] between NMPs or between NMPs and protein residues. A confirmation of that interpretation is given by the presence of that peak only in FFPE liver.…”

Section: Discussionmentioning

confidence: 99%

A Novel HPLC-Based Method to Investigate on RNA after Fixation

Fattorini

Forzato

Tierno

et al. 2020

IJMS

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RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

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“… 11 On the cellular level, FA exerts protein toxicity by modifying the functional components in vital cells, thereby leading to cellular dysfunction. 12 , 13 On the molecular level, FA can react with free thiol and amine groups on protein or DNA, which is followed by the formation of irreversible FA-adducts, 14 as well as FA-catalyzed cross-links of DNA and/or proteins. 15 Nevertheless, there are many issues about the toxicity mechanism of FA that need to be addressed.…”

Section: Introductionmentioning

confidence: 99%