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RNA. 2016 Jan;22(1):155-61. doi: 10.1261/rna.054999.115. Epub 2015 Nov 13.

Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

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Department of Chemistry, The City College of New York, The City University of New York, New York, New York 10031, USA Biochemistry Ph.D. Program, The City University of New York Graduate Center, New York, New York 10016, USA.


Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency.


5′ adenylylation; RNA ligase; RNA-seq; preadenylylation; sequencing adapters; small RNA

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