Photocaged 5-(Hydroxymethyl)pyrimidine Nucleoside Phosphoramidites for Specific Photoactivatable Epigenetic Labeling of DNA

Abstract

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.

Quick Access to Nucleobase-Modified Phosphoramidites for the Synthesis of Oligoribonucleotides Containing Post-Transcriptional Modifications and Epitranscriptomic Marks

“…However, they may be useful for controlling oligonucleotide hybridization when photolabile substituents such as the 2-nitrobenzyl group are used. 28 We obtained photoactivable derivative 8b in 71% yield by alkylation of thymidine phosphoramidite with 2-nitrobenzyl chloride ( Table 1 ).…”

Homologues of epigenetic pyrimidines: 5-alkyl-, 5-hydroxyalkyl and 5-acyluracil and -cytosine nucleotides: synthesis, enzymatic incorporation into DNA and effect on transcription with bacterial RNA polymerase

“…The quantification was performed with 6-fluorescein-labelled primers or GelRed staining as these methods provide a good balance between accuracy and ease of preparation, avoiding potentially dangerous manipulation with radiolabeled DNA. 31,32,36 The more bulky propyl modification on U also had a modest stimulatory effect (128%) while on C it moderately decreased transcription efficiency (74%). The 1-hydroxyethyl modification on U had no apparent effect (U She , ca.…”

“…30 Later on, we developed transcription switches based on photocaging and release of 5hmU or 5hmC in DNA. 31,32 Furthermore, there are even some examples of very rare natural pyrimidine DNA nucleobases bearing even more bulky modifications, e.g. glycine, 2-aminoethyl 33 and several types of conjugates of 5hmU or 5hmC with glucose, 34,35,36 amino acids, amines etc.…”

An Improved Approach for Practical Synthesis of 5-Hydroxymethyl-2′-deoxycytidine (5hmdC) Phosphoramidite and Triphosphate

“…Afterwards, acetyl (2) [22], cyclic carbamate (3) [23], and t-butyldimethylsilyl (4) [24] have also been employed as protecting groups. More recently, 2-nitrobenzyl-protected 5hmdC phosphoramidite (5) has enabled photocaged epigenetic labeling of DNA fragments at specific positions [25]. Currently, cyanoethyl-protected 5hmdC phosphoramidite (1) is most widely used for automated DNA synthesis and available from commercial suppliers.…”