Although conventional Tetramisole hydrochloride siRNAs are 19 nts duplexes with two nucleotides at 39 overhangs, our data demonstrated that 23 nts siRNAs with dTdT as the 39 overhangs displayed most pronounced efficacy. The base pairing at 2�C8th nucleotides of miRNA is thought to be a key feature for the successful regulation of gene expression. Likewise, base pairs formed by the 59 end of siRNA antisense strand are important for siRNA-target binding and cleavage. After the cleavage of dsRNA to short 21�C23 nts siRNA by Dicer, the RISC complex recognize and unwind the siRNA, then target and cleave the complementary mRNA by the binding of the 59 end of antisense strand to target mRNA. In addition to the importance of traditional ����seed region����, the results of our siRNA mutation experiment suggested that the binding of the 39 region of the TATA-box-targeting siRNAs to target sequences was more essential for their activating function. The results above may be due to the different thermodynamic properties of the binding of RNA-RNA and RNA-DNA hybrids. RNA/RNA duplex is more stable than DNA/DNA double helix and RNA/DNA hybrid duplex with the same nearest-neighbor sequence. Therefore, the effective binding of siRNAs to the TATA-box in gene promoter needs more complementary matches. This might explain the failing try of mutating the 39 ends of Piperacillin Sodium invalid siRNAs into nuclear import sequence to strengthen their potency, which also weakened the binding between siRNAs and the target promoter sequences. Moreover, this observation may attribute to the different mechanisms of action among miRNAs, the traditional repressive siRNAs and the activating siRNAs. The differences are mainly due to three ways: first, the target of miRNAs and the traditional repressive siRNAs is RNA, while that of activating siRNA is DNA; second, the location for action of miRNAs and the traditional repressive siRNAs is in the cytoplasm, while activating siRNA regulate gene expression in the nucleus; third, the functional regulating components of miRNAs and the traditional repressive siRNAs are mainly AGOs and GW182, while the associated proteins for activating siRNAs may also include general transcription factors such as Pol II, TBP, and TFIIB etc., besides the AGO proteins.