2024-03-28T12:59:08Zhttps://www.tdx.cat/oai/requestoai:www.tdx.cat:10803/3793072017-09-03T21:31:23Zcom_10803_1col_10803_131463
nam a 5i 4500
RNA
ARN
Regulació genètica
Regulación genética
Genetic regulation
Oligonucleòtids
Oligonucleótidos
Oligonucleotides
Modulation of the RNAi pathway by chemically modified siRNA molecules
[Barcelona] :
Universitat de Barcelona,
2016
Accés lliure
http://hdl.handle.net/10803/379307
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AAMMDDs2016 sp ||||fsm||||0|| 0 eng|c
Alagia, Adele,
autor
1 recurs en línia (281 pàgines)
Tesi realitzada a l'Institut de Química Avançada de Catalunya (IQAC-CSIC)
Tesi
Doctorat
Universitat de Barcelona. Facultat de Química
2015
Universitat de Barcelona. Facultat de Química
Tesis i dissertacions electròniques
Eritja i Casadellà, Ramon,
supervisor acadèmic
Terrazas Martínez, Montserrat,
supervisor acadèmic
Grandas Sagarra, Anna,
supervisor acadèmic
TDX
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.
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