# Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

## TL;DR

This paper introduces a new method for efficiently creating adenylated DNA and RNA adapters for microRNA studies using T4 RNA ligase 1, which is faster and avoids purification steps.

## Contribution

A novel, efficient, and purification-free enzymatic method for adenylating DNA and RNA adapters using T4 RNA ligase 1.

## Key findings

- The method achieves ~100% adenylation efficiency for both DNA and RNA adapters.
- PEG addition reduces adenylation speed differences between DNA and RNA and among terminal nucleotides.
- Running reactions to completion prevents bias caused by adenylation speed differences.

## Abstract

MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences.