Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

Abstract

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93 AE 2 % of EGFP mRNA at a concentration of 10 ng mL À1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55 AE 27 ng of mRNA from 3.1 mg of total RNA, which was not statistically different from the amount recovered using Dynabeads mRNA DIRECT Kit (89 AE 24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.

Strategies towards submicron size and high performance magnetic PGMA@Fe3O4@SiO2-COOH microspheres with biological application

“…The Fe 3 O 4 shell in the composite microspheres is mainly synthesized by an in situ coprecipitation method, which has the same mechanism as the traditional coprecipitation method, i.e., rstly polymer microspheres were mixed with Fe 2+ and Fe 3+ solutions and then a fast coprecipitation reacted under a special environment with high temperature and pH range of 8~14 [21]. This method was widely used and has developed into products like Dynabeads ® [22]. However, up to now, most of the studies of in situ coprecipitation magnetic polymer microspheres were based on micron-sized polymer spheres [23][24][25][26], and there are few studies focused on submicron and monodispersed polymer spheres, also the bonding mechanism of Fe 3 O 4 nanoparticles and polymer microspheres has not been well explained.…”