Reprogramming the assembly of unmodified DNA with a small molecule

The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA ‘alphabet’ by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials

Preparation of Splicing Competent Nuclear Extracts

Splicing components play an essential role in mediating accurate and efficient splicing. The complexity of the spliceosome and its regulatory networks increase the difficulty of studying the splicing reaction in detail. Nuclear extracts derived from HeLa cells provide all of the obligatory components to carry out intron removal in vitro. This chapter describes the large-scale preparation of nuclear extract from HeLa cells. © 2014 Springer Science+Business Media, LLC

Global and Quantitative Profiling of Polyadenylated RNAs Using PAS-seq

mRNA alternative polyadenylation (APA) has been increasingly recognized as a widespread and evolutionarily conserved mechanism for eukaryotic gene regulation. Here we describe a method called poly(A) site sequencing that can not only map RNA polyadenylation sites on a transcriptome level but also provide quantitative information on the relative abundance of polyadenylated RNAs. This method has been successfully used for both global APA analysis and digital gene expression profiling