Synthesis and Purification of N<sup>3</sup>‐Methylcytidine (m<sup>3</sup>C) Modified RNA Oligonucleotides

Mathivanan, Johnsi; Du, Jinxi; Mao, Song; Zheng, Ya Ying; Sheng, Jia

doi:10.1002/cpz1.307

Cited by 3 publications

(3 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For this work, we modified this sequence to contain native cytidines or modified cytidines. We previously reported the synthesis of m 3 C and m 4 C phosphoramidites ( 24 , 25 ) as well as solid phase synthesis of RNA oligonucleotides containing these modifications ( 31 , 32 ). Here, we used this optimized oligonucleotide synthesis procedure to synthesize a 22 nt native RNA as well as RNA containing one or two m 3 C, m 4 C and 2′-O-Me modifications (see Methods).…”

Section: Resultsmentioning

confidence: 99%

“…For modified RNA oligomer synthesis, 2-Thio uridine CE phosphoramidite, 4-thio uridine phosphoramidite and 2′- O -methyl cytidine phosphoramidite were purchased from ChemGenes. N4-methyl-cytidine phosphoramidite and N4-benzyl-N3-methyl-cytidine phosphoramidite were synthesized in the laboratory based on the protocol we developed earlier ( 24 , 25 ). Oligonucleotide sequences used in this study are provided in Supplementary Table S1 .…”

Section: Methodsmentioning

confidence: 99%

“…All modified RNA oligonucleotides were synthesized in-house by standard solid phase synthesis using an automated Oligo-800 DNA synthesizer ( 24 , 25 ). After synthesis, oligonucleotides were cleaved and deprotected using AMA (a mixture of ammonium hydroxide and methylamine solution, 1:1) by heating the sample at 65°C for 45 min.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Todkari,

Chandrasekaran,

Punnoose

et al. 2023

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

There are >170 naturally occurring RNA chemical modifications, with both known and unknown biological functions. Analytical methods for detecting chemical modifications and for analyzing their effects are relatively limited and have had difficulty keeping pace with the demand for RNA chemical biology and biochemistry research. Some modifications can affect the ability of RNA to hybridize with its complementary sequence or change the selectivity of base pairing. Here, we investigate the use of affinity-based DNA nanoswitches to resolve energetic differences in hybridization. We found that a single m3C modification can sufficiently destabilize hybridization to abolish a detection signal, while an s4U modification can selectively hybridize with G over A. These results establish proof of concept for using DNA nanoswitches to detect certain RNA modifications and analyzing their effects in base pairing stability and specificity.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Todkari,

Chandrasekaran,

Punnoose

et al. 2023

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

Synthesis of N³‐Methyluridine‐ and 2′‐O‐Alkyl/2′‐Fluoro‐N³‐Methyluridine‐Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides

Sahoo,

Gore

2024

Current Protocols

View full text Add to dashboard Cite

In this article, we describe the synthesis of N3‐methyluridine (m3U) and 2′‐O‐alkyl/2′‐fluoro‐N3‐methyluridine (2′‐O‐alkyl/2′‐F‐m3U) phosphoramidites as well as their incorporation into a 14‐mer DNA and RNA oligonucleotide sequence. Synthesis of the 2′‐O‐alkyl‐m3U phosphoramidite starts with commercially available uridine to achieve a tritylated m3U intermediate, followed by 2′‐O‐alkylation and finally phosphitylation. Synthesis of the 2′‐F‐m3U phosphoramidite is obtained from a commercially available 2′‐F‐uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2′‐position of m3U that can be employed in numerous nucleic acid–based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR‐Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Synthesis of 2′‐O‐alkyl‐N3‐methyluridine analogs and their corresponding phosphoramiditesAlternate Protocol 1: Synthesis of 2′‐O‐TBDMS‐N3‐methyluridine and its phosphoramiditeAlternate Protocol 2: Synthesis of 2′‐fluoro‐N3‐methyluridine and its phosphoramiditeBasic Protocol 2: Solid‐phase synthesis of N3‐methyluridine‐modified DNA and RNA oligonucleotides

show abstract

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

et al. 2022

View full text Add to dashboard Cite

Herein, we report a straightforward one-step procedure for modifying N -nucleophilic groups in the nucleobases of commercially available nucleoside phosphoramidites. This method involves the deprotonation of amide groups under phase-transfer conditions and subsequent reaction with electrophilic molecules such as alkyl halides or organic isocyanates. Using this approach, we obtained 10 different classes of modified nucleoside phosphoramidites suitable for the synthesis of oligonucleotides, including several noncanonical nucleotides found in natural RNA or DNA (e.g., m 6 A, i 6 A, m 1 A, g 6 A, m 3 C, m 4 C, m 3 U, m 1 G, and m 2 G). Such modification of nucleobases is a common mechanism for post-transcriptional regulation of RNA stability and translational activity in various organisms. To better understand this process, relevant cellular recognition partners (e.g., proteins) must be identified and characterized. However, this step has been impeded by limited access to molecular tools containing such modified nucleotides.

show abstract

Synthesis and Purification of N³‐Methylcytidine (m³C) Modified RNA Oligonucleotides

Cited by 3 publications

References 45 publications

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Synthesis of N³‐Methyluridine‐ and 2′‐O‐Alkyl/2′‐Fluoro‐N³‐Methyluridine‐Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides

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

Contact Info

Product

Resources

About

Synthesis and Purification of N3‐Methylcytidine (m3C) Modified RNA Oligonucleotides

Cited by 3 publications

References 45 publications

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Resolving altered base-pairing of RNA modifications with DNA nanoswitches

Synthesis of N3‐Methyluridine‐ and 2′‐O‐Alkyl/2′‐Fluoro‐N3‐Methyluridine‐Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides

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

Contact Info

Product

Resources

About

Synthesis and Purification of N³‐Methylcytidine (m³C) Modified RNA Oligonucleotides

Synthesis of N³‐Methyluridine‐ and 2′‐O‐Alkyl/2′‐Fluoro‐N³‐Methyluridine‐Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides