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232 Cheng and ChangFig. 4. Sequence-specific features for the rational design of potential siRNAs. Themature siRNA is a 21–23-nt dsRNA that contains a 19-nt duplexed region, symmetrical2-nt 3′-overhangs, and 5′-phosphate (P) and 3′-hydroxyl (OH) groups. The positionsof each nucleotide in the 19-nt duplexed region of the sense strand are numbered. Onthe basis of recently established design rules, an effective siRNA has high stability atthe 5′-terminus of the sense strand, and lower stability at the 5′-antisense terminus andat the cleavage site. In addition, the sequence-specific preferences at the following positionson the sense strand are important including the presence of a G (C) at position 1,an A at position 3, an U at position 10, and an A (U) at position 19.pMSCVpuro vector is inserted into the ClaI/HindIII-digested pHsH1, pHsU6,pMmH1, and pMmU6 vectors.3.2. Molecular Characteristics of Designed and SelectedRNAi-Targeting SequencesThe efficiency of RNAi-based gene silencing is primarily dependent on theeffectiveness and specificity of the RNAi-targeting sequences. To obtain theeffective siRNAs, it is necessary to design, synthesize, and screen several differentRNAi-targeting sequences from a particular gene. Systematic analyses of thespecific features from the effective siRNAs reveal that siRNA might havesequence-specific characteristics associated with its functionality. These molecularcharacteristics generally include low-to-medium G/C content (30–50%), highinternal stability at the sense strand 5′-terminus, low internal stability at the sensestrand 3′-terminus, absence of internal repeats or palindromes, and base preferencesat the sense strand positions 1, 3, 10, and 19 (see Fig. 4) (39–43).1. Retrieve the nucleotide sequence of any gene from the National Center forBiotechnology Information (NCBI) nucleotide database (GenBank; http://www.ncbi.nlm.nih.gov/).2. Screen any 19-nt sequence (see Note 5) within the coding region and 3′-untranslatedregion that fulfills the aforementioned sequence-specific characteristics and inparticular does not contain stretches of four or more consecutive As and Ts.3. Select any 19-nt sequence containing more than three mismatches to any othergene and also avoid any known single nucleotide polymorphisms by searching the