A systematic study on the influence of thermodynamic asymmetry of 5'-ends of siRNA duplexes in relation to their silencing potency

Abstract

siRNA molecules possess high potential as molecular tools and can be used as effective therapeutics in humans. One of the key steps in the action of these molecules is the choice of antisense strand by the RNA-induced silencing complex (RISC). To explain this process, we verified the theory which states that antisense strand selection is based on the thermodynamically less stable 5' end of siRNA. Based on the studies presented herein, we observed that for the tested siRNA duplexes, the difference in the thermodynamic stability of the terminal, penultimate and pre-penultimate pairs in the duplex siRNA is not the dominant factor in antisense strand selection. We found that both strands in each tested siRNA molecule are used as an antisense strand. The introduction of modified nucleotides, whose impact on the thermodynamic stability of siRNA duplexes was studied, results in changes in antisense strand selection by the RISC complex. The presence of a modified residue often caused predominant selection of only one antisense strand which is at variance with the theory of siRNA strand bias.

Figure 1

Sequences of siRNA duplexes with defined 5′-end thermodynamic stability. The duplexes with thermodynamically diversified 5′ ends are shown in panels (A) A1/A2 and (B) B1/B2. The duplexes with equal thermodynamic stability of 5′ ends are shown in panels (C) C1/C2 and (D) D1/D2. A dot near a specific strand indicates the strand which should be selected as antisense strand according to the theory of a less stable 5′-end.

Figure 2

Scheme of HeLa cell line experiments. Each siRNA duplex was examined in two individual experiments. The siRNA molecule was co-transfected with two plasmids. One of these contained an insert with a complementary sequence to one of the siRNA strands, while the second plasmid contained an insert with a complementary sequence to the second strand of the siRNA duplex. After 24 h total RNA isolation, cDNA synthesis and qPCR analysis were performed.

Figure 3

The change of ZsGreen1 relative normalized expression induced by siRNAs: A1/A2, B1/B2, C1/C2, D1/D2. Each bar represents the mean value ± standard deviation (SD) of n = 3–5. *Indicates statistical significance at a level of P < 0.05; ***indicates statistical significance at a level of P < 0.01.

Figure 4

Schematic representation of siRNA duplexes and modified nucleosides used in the thermodynamic studies: (A) Model siRNA duplex used in UV melting experiments, X-Y correspond to unmodified or modified base pairs or mismatches, (B) structure of 2-thiocytidine, 2-thiouridine, and 4-thiouridine.

Figure 5

Results of UV melting experiments. ΔΔG°₃₇ corresponds to the difference in free energy between a model duplex and a duplex containing a mismatch or modified nucleotide. Positive bar values indicate thermodynamic destabilization and negative bar values indicate thermodynamic stabilization. Underlined base pairs mean references for which a free energy difference was determined. (A) The influence of mismatches (B) the influence of s²C substitution, (C) the influence of s²U substitution, (D) the influence of s⁴U substitution.

Figure 6

Hydrogen bonding between G-C, G-s²C, A-U, A-s²U, and A-s⁴U.

Figure 7

The change of ZsGreen1 relative normalized expression induced by siRNA molecules containing modified nucleotides. The activity of: (A) A3/A4 duplex with 2-thiouridine (s²U) at pre-penultimate position of A3 strand, (B) B3/B4 duplex with 2-thiocytidine (s²C) at penultimate position of B3 strand, (C) C3/C4 duplex with mismatch C-A at pre-penultimate position of C3 strand, (D) C5/C6 duplex with 2-thiocytidine (s²C) at pre-penultimate position of C6 strand, (E) C7/C8 duplex with mismatch C-A at pre-penultimate position of C7 strand, (F) D3/D4 duplex with 2-thiouridine (s²U) at pre-penultimate position of D4 strand, (G) D5/D6 duplex with mismatch G-U at penultimate position of D5 strand, (H) D7/D8 duplex with s⁴U-G mismatch at penultimate position of D8 strand, and (I) D9/D10 duplex with 4-thiouridine (s⁴U) at penultimate position of D10 strand. Each bar represents the mean ± standard deviation (SD) of n = 3–5. *Indicates statistical significance at a level of P < 0.05; ***indicates statistical significance at a level of P < 0.01.

Figure 8

Schematic representation of the effect of mismatches and modified nucleotides on the thermodynamic stability of siRNA model duplexes in terminal, penultimate and pre-penultimate position. In addition, the pairs that were introduced to the full-length siRNA and used in HeLa cell line experiments were marked and the impact of the strand selection by RISC complex were noted.