Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures

A Globin Blocker to Increase Sequencing Efficiency for QuantSeq 3' mRNA-Seq in Porcine Blood

Blood is an extremely valuable source to investigate animal biology, including whole genome gene expression. The efficiency of gene expression profiling is hampered by the high proportions of hemoglobin (HB) mRNA of erythrocytes, which decreases the resolution of QuantSeq 3' mRNA-sequencing in blood. Here, we used different concentrations of a globin blocker (GB), which includes a HB-specific oligonucleotide mix, as a novel method to decrease sequencing of HB RNA. While reads for HBA and HBB accounted for 19.4 and 36.9% of total reads in non-GB samples, GB under optimal concentrations reduced HBA and HBB reads on average to 8.7 and 2.3% of total reads, respectively. The number of genes that could be reliably detected was of approximately 2,200 genes greater in GB samples than in non-GB samples. In conclusion, the use of this GB procedure increases the resolution of gene expression data from QuantSeq in porcine blood samples.</p

A comprehensive map of alternative polyadenylation in African American and European American lung cancer patients.

Deciphering the post-transcriptional mechanisms (PTM) regulating gene expression is critical to understand the dynamics underlying transcriptomic regulation in cancer. Alternative polyadenylation (APA)-regulation of mRNA 3\u27UTR length by alternating poly(A) site usage-is a key PTM mechanism whose comprehensive analysis in cancer remains an important open challenge. Here we use a method and analysis pipeline that sequences 3\u27end-enriched RNA directly to overcome the saturation limitation of traditional 5\u27-3\u27 based sequencing. We comprehensively map the APA landscape in lung cancer in a cohort of 98 tumor/non-involved tissues derived from European American and African American patients. We identify a global shortening of 3\u27UTR transcripts in lung cancer, with notable functional implications on the expression of both coding and noncoding genes. We find that APA of non-coding RNA transcripts (long non-coding RNAs and microRNAs) is a recurrent event in lung cancer and discover that the selection of alternative polyA sites is a form of non-coding RNA expression control. Our results indicate that mRNA transcripts from EAs are two times more likely than AAs to undergo APA in lung cancer. Taken together, our findings comprehensively map and identify the important functional role of alternative polyadenylation in determining transcriptomic heterogeneity in lung cancer