Optimization of N-hydroxysuccinimide ester coupling with aminoallyl-modified RNA for fluorescent labeling

Abstract

Site-specific fluorescent labeling of RNA is crucial for obtaining the structural and dynamic information of RNAs by fluorescence techniques. Post-synthetic modification of RNA based on N-hydroxysuccinimide (NHS) coupling reaction is an economic, efficient and simple strategy to introduce fluorophore to samples. However, this strategy are not that frequently used in RNA molecules, and the reported reaction conditions and yields varied among different systems. This study results mainly focused on screening the reaction conditions (reactants concentrations, dimethylsulfoxide concentration, solution conditions, pH and reaction time) between NHS-linked fluorophore and aminoallyl-RNA (aa-RNA) to optimize the yield of fluorescent RNA up to 55%, doubled the initial yield. What’s more, as low as one tenth of fluorescent reagent was used in our protocol compared with the reported protocols, greatly reducing the experimental cost. The protocol can be applied as a general guide potentially for RNA labeling by NHS-ester coupling reaction.

Intracellular RNA Labeling Technologies for the Analysis of RNA Biology†

“…Additionally, the length of the synthesized RNA is limited, and the functional group on the nucleoside could affect the recognition and elongation efficiency of the enzyme. Connective hubs, such as N ‐hydroxysuccinimide (NHS) esters and sulfuryl fluoride (SO 2 F 2 ), [ 34‐35 ] could chemo‐selectively bioconjugate a fluorophore, biotin or other functional groups to the end of the nucleic acid in vitro , which could be further applied for imaging, enrichment, or drug screening. Recently, a genetically encoded chemical labeling strategy called GECX‐RNA was developed for the site‐specific crosslinking of RNA and protein.…”

Amine‐to‐Azide Conversion on Native RNA via Metal‐Free Diazotransfer Opens New Avenues for RNA Manipulations

“…In summary, our new approach for selective amine‐to‐azide conversions on RNA targets containing primary amino functionalities paves the way to RNA bioconjugation approaches that make use of the most efficient biorthogonal reactions known to date for azido compounds. In this context, we point out that the direct labeling of a sterically hindered primary amino group by using active ester[ 65 , 66 , 67 ] or isocyanate [68] reagents is usually troublesome and inferior to the here introduced straightforward two‐step procedure because of low yields and tedious optimization of pH‐ and salt‐dependent reaction conditions. We are confident that the new diazotransfer reaction with the FSO 2 N 3 reagent will significantly expand the repertoire of bioorthogonal RNA bioconjugation chemistry, enabling exciting new biochemical applications ranging from the chemically controlled spatial and temporal activation of RNAs to the direct manipulation of metabolically labeled RNAs.…”