SiRNAs molecules

As an alternative to cationic lipids, the potential of anionic lipids for DNA delivery has been investigated. Because of their negative charge, DNA or siRNA molecules are very inefficiently entrapped by anionic lipids alone. In the presence of cations such as K+, Na+, or Ca2+, the complex formation of such anionic lipids and nucleic acids can be enhanced. The resulting ternary complexes of DNA, anionic lipids, and divalent calcium ions have been reported to transfect mammalian cell lines efficiently [33]. Despite this, there has only been limited progress with these anionic lipid DNA delivery systems, a fact that may be attributed, in part, to the poor association between DNA molecules and anionic lipids, caused by electrostatic repulsion between these negatively charged species. 

The introduction of synthesized siRNA molecules or the expression of short hairpin RNA (shRNA) precursor structures is important in mammalian RNAi experiments. However, the introduction of dsRNA longer than 30 nucleotides (in mammalian cells) causes an apoptotic response. This interferon response is initiated by... 

FIGURE 9.1 Target mRNA silencing. A simple schematic showing how siRNA molecules are involved in the RNA interference process [9],... 

Several groups have performed various experiments to determine the preferred characteristics when designing siRNA molecules and the important considerations when selecting a target sequence. For example, designing a specific and potent siRNA is very important for any RNA interference study. The benefits of generating potent siRNAs may include the following [1] ... 

Because the physical attributes of siRNA molecules are important for a successful RNAi experiment, one should use siRNA molecules at their lowest effective amount [11], As the reader will learn in the next several sections, the suggested guidelines for designing a specific and potent siRNA have been exploited and automated using a variety of in silico methods. It should also be noted that these are simply guidelines, and they may still lead to off-target effects or other undesired experimental results. 

Many design rules from various groups have been created to optimize the specificity of a siRNA molecule (fig. 9.2). Many of these rules have evolved as the RNAi technology has become more prevalent in mRNA expression studies. How an siRNA duplex sequence is related to its target mRNA sequence can be illustrated with an example target region from Lamin A/C [7]. 

Because the antisense siRNA strand interacts with the mRNA of the target gene, one should find a region on the target sequence that interacts well with the antisense siRNA strand, and one must also use suitable design rules for creating a specific and stable siRNA molecule for a successful RNAi experiment. 

The first set of rules for designing potent siRNA molecules was established by Tuschl s group (http //www.rockefeller.edu/labheads/tuschl/sima.html [12]). Many current siRNA designers and software programs use these rules to help scientists generate potent siRNA molecules. The rales are simple to understand and have been well summarized by Ding and Lawrence [13],... 

More recently, a few academic research labs and companies have examined the properties of effective siRNA molecules and created algorithms to help them extend some of the simple rules first established by Tuschl s group. Ding and Lawrence [13] expanded these rules to include secondary structure and accessibility prediction in their program called SiRNA. Their basic siRNA design steps include the following ... 

Although designing a potent siRNA molecule is important for many RNAi experiments, it may also be important to consider the attributes of the target gene sequence. 

Once the rules of siRNA design and target selection became more accepted, in silico siRNA prediction came to the fore. Bioinformaticists now use empirical and theoretical design rules to generate siRNA molecules as described earlier. However, because these design approaches use different algorithms, their siRNA design... 

Although there are a number of public siRNA design tools available on the Internet, many siRNA supply companies offer free siRNA design tools for their customers. These companies hope customers visit their Web sites, use their design tools, and generate siRNA molecules that can be ordered directly from their Web site. This Web site approach has been adopted by several companies such as Ambion, Dharmacon, and Invitrogen (see table 9.3). Because commercial Web sites may use different siRNA... 

One bioinformatics company that offers specialized siRNA software tools is called Ocimum Biosolutions. Their software, iRNAwiz, provides an environment for the design of successful siRNA molecules and is composed of several components. These components include a siRNA Search tool, a BLAST tool, a Motif Search tool, a Stemloop search, and a Statistical Analysis tool [32]. They claim the combination of these tools will result in the design of siRNA molecules with high efficiency. 

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