NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Determination of enrichment factors for modified RNA in MeRIP experiments.

In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry 32P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

Absolute Quantification of Noncoding RNA by Microscale Thermophoresis.

Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization-based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA-Seq-based quantification approaches, strongly suggesting a bias due to tRNA modifications in the latter. Further applications include the quantification of rRNA as well as of polyA levels in cellular RNA.

Effects of oxygenation on the intercalation of 1,10-phenanthroline-5,6/4,7-dione between DNA base pairs: a computational study.

The effects of oxygen in positions 4,7 and 5,6 of phenanthroline when this ligand intercalates between guanine-cytosine and adenine-thymine DNA base pairs (GC/CG and AT/TA) have been studied at the M06-2X/6-31+G(d,p) level of calculation. We focused on the changes in the structure, stabilization and energy contributions in the analysis of the interaction. The obtained trends in stabilization are explained by a model including repulsive Pauli (ΔEPauli) contributions, and attractive dispersion (ΔEdisp), orbital (ΔEorb) and electrostatic (ΔEelstat) contributions to energy. When no solvation is considered, the intrinsic ΔEelstat contribution results are crucial for the stabilization of the system. However, the inclusion of the solvation energy ΔEsolv can reverse the final stability trend of the systems becoming, thus, the driving force of the process. Therefore, the solvent will have a relevant influence in the potential cytotoxicity of the intercalation drugs.