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

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.

Modified forms of easyCLIP

“…In a crystal structure, T4 RNA ligase makes RNA contacts with the 3’OH-providing substrate, but does not contact the RNA bases of the 5’PO 4 -providing substrate 3 , consistent with its requirement for RNA bases in the 3’OH-providing substrate and not in the 5’PO 4 -providing substrate 4 . RNA ligase can also pre-adenylylate DNA oligos efficiently as long as the 5’ base is not G 5 . These facts suggest the pre-adenylylated DNA oligo would be competitive with the 5’RNA bases form, but do not prove either would necessarily work better in the context of CLIP.…”

“…The easyCLIP method, describing both a CLIP-seq library construction method (Figure 1a) and a UV protein-RNA cross-link rate measurement method, was presented in paper 1 claiming the following strengths: (1) it was an easy, quick and reliable method to produce normal CLIP-seq libraries; (2) it provided troubleshooting information based on the fluorescent markers on adapters; (3) it quantified the absolute rate of protein-RNA cross-linking; (4) it enabled distinguishing RBPs from non-RBPs on the basis of combining cross-link rate and CLIP libraries; (5) on the same basis, it enabled distinguishing "specific" RNA interactions, vs those of a non-RBP.…”

Determination of absolute expression profiles using multiplexed miRNA analysis

“…Amplification bias has been minimized by optimizing reaction conditions to suppress ligation bias [ 32 , 33 ] and by optimizing probe design via thermodynamic analysis. Amplification bias can be determined by analyzing the spread in the fluorescent intensity across miRNA.…”

A TBP-independent mechanism for RNA Polymerase II transcription

“…Barcoded DNA linkers were riboadenylated in-house as followed in Song et al (115). Briefly, adenylation reactions were performed in a reaction mixture (10 µL) containing 0.8 ug of barcoded DNA linker, 1X T4 RNA ligase buffer, 2 mM ATP, 35% PEG, and 300 U of T4 RNA Ligase 1, incubated at 37 °C for 8 hours, followed by 15 minutes of heat inactivation at 65 °C.…”