RESUMEN

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.

Asunto(s)

Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/síntesis química , Celulosa/química , Ingeniería Química/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Cromatografía de Afinidad , Técnicas In Vitro , Datos de Secuencia Molecular , Oligonucleótidos/genética , ARN Catalítico/química

RESUMEN

N-Alkyl-N-nitrosoamides undergo competitive reactions whose rates are dependent upon the interplay of a number of factors. There already exists a significant body of work delineating the effects of pH on the partitioning of the nitrosoamides along their deaminative (-N(2)) and denitrosative (-"NO(+)") pathways. In this paper, the issue of pH dependence is discussed with particular attention to nitrosoamide decompositions in nonaqueous media. The role of the acidity of the medium in the partitioning of the nitrosoamide between deamination and denitrosation and in the choice of deaminative pathways is revisited. In nonaqueous media under near-neutral conditions, the partitioning's pH dependence is evidently accompanied by a sensitivity to structural features in the nitrosoamide. Thus, diminution of steric crowding around the N-nitroso moiety as well as the presence of strongly electron-withdrawing acyl units (i.e., those derived from strong acids, e.g., tosyl and trifyl) increase the relative yield of amides by encouraging the denitrosative pathway. A mechanism for thermal denitrosation of nitrosoamides under near-neutral conditions is proposed in which rapid protonation at the acyl O rather than slow protonation at the amidic N is the first step in the reaction profile. A rate-limiting, bimolecular reaction between the O-conjugate acid and adventitious nucleophiles at the nitrosyl group then occurs followed by rapid tautomerization to amide.