Chemical Modifications Enable Unique RNAi Applications.
In a cell, RNA is made by the transcription of DNA by intracellular enzymes. RNA can also be made in vitro by enzymes like T7 RNA polymerase. A third option for making RNA exists - chemical synthesis - which differs from in vivo and in vitro RNA production because it does not use enzymes to make RNA. Instead, chemical synthesis works by adding one RNA base at a time in repeated cycles. In this way, short single-strand RNA molecules, or RNA oligonucleotides, of exactly defined sequence and length are made.
Since 1995 Dharmacon has been a leader of RNA synthesis technology through the development of a novel RNA synthesis chemistry that is so advanced we patented it! This unique chemistry uses a new class of silyl ethers to protect the 5'-hydroxyl with a unique acid-labile orthoester protecting group on the 2'-hydroxyl [1, 2]. The chemical name of this protecting group is 2´-bis(acetoxyethoxy)-methyl ether, but we commonly refer to it as “2'-ACE”. To learn more, read our 2'-ACE RNA Synthesis Chemistry Technical Note which explains our patented RNA synthesis chemistry in greater detail.
The innovative properties of 2´-ACE technology enable routine synthesis of siRNA in high yield and of exceptional quality. This is because 2´-ACE phosphoramidites allow high coupling efficiencies resulting in high quality RNAs and siRNAs. The purity levels from this synthesis technology, typically 80-85% for unmodified siRNA, are routinely higher than other methods (e.g. 2'-tert-Butyldimethylsilyl ether, TBDMS). Other advantages of 2'-ACE protecting groups are water solubility, nuclease resistance, synthesis of strong secondary structures, and a final acid deprotection that is mild, fast, and requires minimal handling.
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Chemical synthesis of siRNAs allows for position-specific incorporation of a wide variety of modifications into your sequence. These modifications can provide siRNA performance advantages for various experimental applications [3, 4].
The simplest approach to increase nuclease resistance of siRNA is to directly modify the internucleotide phosphate linkage. Replacement of a non-bridging oxygen with sulfur (phosphorothioate, PS) has been extensively used to improve nuclease resistance of siRNAs .
Modifications of the 2'-position of the ribose can indirectly improve nuclease resistance of the internucleotide phosphate bond while increasing duplex stability (Tm). This modification has also been shown to provide protection from immune activation.
A combination of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-Methyl, 2'-Fluoro, 2'-Hydro) has often created highly stable siRNAs for applications in vivo .
The transient nature of siRNA silencing is primarily due to the continuous dilution of intracellular siRNA through cellular division and nucleic acid degradation. Chemical modifications help to ensure delivery of more intact siRNAs into the cell, and subsequently stabilize the delivered siRNA for proper RISC recruitment. For example, prolonged silencing has been achieved in cell culture using 2'-OMe- modifications within the siRNA .
One major challenge for in vivo siRNA is undesired activation of the innate immune system. The immunostimulatory effects of dsRNA are mediated primarily through three Toll-like receptors (TLR3, TLR7, and TLR8) and by proteins such as retinoic acid inducible protein (RIG-1) , oligoadenylate synthetase (OAS), dsRNA-responsive kinase (PKR), and melanoma differentiation associated protein (MDA-5) .
Rendering siRNAs unrecognizable to these immunostimulatory receptors through chemical modifications has been shown to significantly decrease siRNA immunogenicity. Modifying specific sequences with 2'-OMe, 2'-F, and 2'-H can effectively reduce TLR7/TLR8 interaction while generally preserving silencing activity [10, 11]. Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effects mediated by TLR3, TLR7, and TLR8 .
When either strand of an siRNA reduces the expression of a gene that is not the intended target - this is undesirable and known as “off-targeting”. There are two ways off-targeting can occur. The siRNA guide strand can target an mRNA transcript from a gene that has a sequence similar to the target gene. A second way off-targeting occurs is through loading of the siRNA passenger strand into RISC, and degradation of mRNA targets that have sequences complementary to the passenger strand. Both of these off-targeting mechanisms usually occur through high degrees of sequence similarity in the seed region, and imperfect matches throughout the rest of the siRNA oligonucleotide. For this reason, these off-targeting events are considered to be microRNA-like in nature .
To prevent either siRNA strand from miRNA-like off-targeting, position-specific modifications are used.
Chemical modification can help solve some of the problems associated with delivery of siRNA into certain cells and animals. Due to the negatively charged phosphate backbones of RNA and its analogues, and the non-polar nature of the lipid bi-layer that makes up the cell membrane, getting siRNA into a cell can be difficult. For systemic delivery of siRNA, various conjugates have been shown to help delivery of siRNA into cells. These modifications include:
These modifications can increase the potential use of siRNA as a systemic drug .
For some siRNA experiments, imaging siRNA can be a useful control for delivery, or to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of the molecules in the experimental model.
Here are multiple ways imaging probes can be used:
The best modification to use in your custom siRNA design depends upon your intended application and can vary with the design of siRNA, specific sequence, and method of delivery. Chemical modifications in RNA synthesis can widen the experimental approaches of RNAi that can be taken. The vast array of chemical modification schemes offered by Dharmacon will greatly extend your research capabilities. If you have questions about chemical modifications for custom RNA synthesis, speak with a scientist on our Technical Support Team.
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Create and order RNA molecules with a wide variety of chemical modifications or learn about our capabilities for long RNA oligos, dye labeling and custom amidites.
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A significant improvement in RNA synthesis technology, 2'-ACE chemistry results in higher yields, greater purity and superior ease of handling.
A new class of silyl ethers is used to protect the 5’-hydroxyl (5’-SIL) in combination with an acid-labile orthoester protecting group on the ...
The dsRNA is cleaved by the RNase-III enzyme, Dicer, into small interfering RNAs (siRNA) that are approximately 21-23 nt.