Modulation of the RNAi pathway by chemically modified siRNA molecules

Abstract

[spa] Para dirigir el silenciamiento génico post-transcripcional, la maquinaria de RNAi explota la formación de pares de bases entre la hebra guía cargado y el ARNm complementario. La proteína Ago2 (Argonauta 2) es la "máquina de cortar" del complejo RISC y dirige la rotura endonucleolítica sólo cuando la hebra guía del siRNA está completamente apareada con su homóloga de ARN. Ago2 es capaz de incorporar una molécula de dúplex de siRNA, desenrolla la doble hélice y mantiene una hebra mientras se descarta la otra cadena. Ago2 cargada con la hebra guía se define "activa" y puede guiar múltiples reacciones de escisión contra los ARNm complementarios. El análisis estructural del proceso de ensamblaje de Ago2 ha llevado a la conclusión de que las primeras interacciones entre el siRNA y la proteina Ago2 se basa en el reconocimiento específico por el dominio PAZ. Por lo tanto, el correcto reconocimiento de dominio PAZ contribuye a la incorporación específica y productiva de los siRNAs en el Ago2. La hebra guía con su extremo 3' protuberante que tiene 2-nt está implicada en la mayoría de los contactos entre la cavidad presente en el dominio PAZ. En principio, las modificaciones en los extremos protuberantes se introdujeron para proteger la integridad del dúplex de ARN. Sólo después de la comprensión de la arquitectura de Ago2, se pensó en la utilización de las modificaciones en los extremos protuberantes para mejorar la potencia y especificidad de los siRNAs. Para explorar las características estructurales críticas para la interacción entre la cavidad PAZ, modificamos los extremos protuberantes de los siRNA con varias modificaciones. Específicamente, 2 unidades de un beta-L-nucleósido como la L-timidina (imagen especular de la timidina), de 2'-desoxiribitol, de GNA (glycerol nucleic acids)- timina y del derivado acíclico L-treoninol se introdujeron a los extremos protuberantes y se midió la potencia de silenciamiento (IC50). Tales modificaciones pueden proporcionar pistas fundamentales sobre el requisito estructural necesario para el reconocimiento y carga de la cadena del dominio PAZ de Ago2.

[eng] To direct post-transcriptionally gene silencing, RNAi machinery exploits the formation of base pairs between the loaded guide strand and the complementary mRNA. The Ago2 protein is the “slicer” effector of the RISC and drives the endonucleolytic cleavage only when the siRNA guide strand is full paired with its RNA counterpart. Ago2 is able to incorporate a duplex siRNA molecule, unwinds the double helix and holds one strand while discarding the other. Ago2 bearing only the guide strand is defined “active” and can guide multiple cleavage reactions against the complementary mRNAs. Structural insights into Ago2 assembly process have speculated that early interactions between the siRNA and the Ago2 relies on specific recognition by the PAZ domain. Thus, proper PAZ domain recognition contributes to the specific and productive incorporation of siRNAs into the Ago2. Interactions between PAZ domain and siRNA molecule are essentially asymmetric. The guide strand with its 2-nt 3’overhang is involved in the majority of the contacts between the PAZ pocket and the siRNA, whereas the passenger strand interacts only with its 5’ end residue. In principle, overhang modifications (i.e. 2’-deoxy units) were just introduced to protect the RNA duplex integrity. Only after the understanding of the Ago2 architecture, overhang modifications were also harnessed to improve the siRNA potency and specificity. The comprehension of the PAZ lodging/dislodging motion during the formation of binary (Ago2 + guide) and ternary complex (Ago2 + guide + mRNA) pointed out the importance of adequate affinity between the guide overhang and the PAZ cleft during the Ago2 multi-turnover cleavage process. Affinity analysis on PAZ/siRNA overhang complex has proved the influence of the overhang presence for efficient binding. SiRNA duplexes with shorter overhang (1-nt) or blunt end have respectively highlighted 85-fold and >5000-fold reduced affinity. Hence, taking advantage of more efficient interactions between the PAZ pocket and the strand bearing the unpaired di-nucleotides structure, structural asymmetric siRNA molecules bearing only the antisense overhang were successful employed to bias the RISC strand selection. Moreover, competition between siRNAs, resulting in preferential incorporation of one siRNA type into the RISC machinery, is influenced by the distinct loading kinetics of siRNA molecules. Thus, the knockdown ability of siRNA mixtures is often compromised due to competition between siRNAs. It also has been reported that the simultaneous transfection of two or more siRNAs causes reduced silencing activity of one siRNA species whereas the potency of the other siRNAs were not affected. Even if siRNA competition effects are essentially produced by the interactions with the Ago2 protein, up to now, no available data about a specific Ago2 domain involvement into the siRNA competition have been described. We have been hypothesized that the PAZ domain, playing an important role in the first steps of the strand loading could be specifically involved in the siRNA competition. Given this background we are questioning how the di-nucleotide unpaired structure can influence the siRNA silencing efficiency and specificity. To explore the structural hallmarks critical for the PAZ pocket interaction, we modified the siRNA overhangs with several modifications. In detail, 2 units of β-L-nucleosides (mirror image L-Thymidine), 2’-deoxyribitol, GNA (glycerol nucleic acids)-Thymine and acyclic L-threoninol were introduced at overhang level and the silencing potency (IC50) was measured. Such modifications may provide fundamental clues on structural prerequisite needed for the PAZ recognition and strand loading into the Ago2.