Research Techniques Made Simple:Small Interfering RNA (siRNA)

Vinod E. Nambudiri, MD MBA1,2, Rachael Clark, MD PhD3, Hans Widlund, PhD MSc3

1. Harvard Combined Dermatology Residency Program, Boston, Massachusetts 2. Department of Internal Medicine, Brigham and Women’s Hospital, Boston, Massachusetts

3. Department of Dermatology, Brigham and Women’s Hospital, Boston, Massachusetts

Processes of RNA Interference• The traditional flow of genetic information is

from DNA to mRNA to protein.• RNA interference uses specifically designed RNA

molecules to reduce the expression of targeted genes.

• Small interfering RNAs (siRNAs) are constructed to be complementary to target mRNA sequences.

• The binding of siRNAs to target mRNAs leads to the cleavage of the mRNAs and thus decreased target protein synthesis.

Processes of RNA Interference• Once introduced into cells, double-stranded

siRNAs are trimmed into 21- to 23-base-pair fragments.

• One strand of the siRNA binds with the proteins of the RNA-induced silencing complex (RISC) in the cellular cytoplasm.

• After complementary base pairing with the target mRNA, the RISC complex is activated and the mRNA transcript is cleaved.

• siRNAs are a mode of posttransciptional gene silencing.

Technical Aspects of siRNA• The target gene’s DNA sequence and corresponding

mRNA transcript are identified. • A series of complementary siRNAs is generated. • Once the siRNAs are synthesized as double-stranded

RNAs, they must then be introduced into the cells of interest via a process known as transfection.

• After the siRNAs are introduced into the cellular cytoplasm, they are able to be processed by cellular enzymes as described above and participate in the process of posttranscriptional gene silencing.

• Quantification of siRNA efficacy can use techniques such as real-time reverse transcription PCR and western blotting.

Summary of siRNA Gene Silencing

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siRNA in Dermatology• Using siRNA to better

understand and treat single-gene defect cutaneous diseases:– Dominant dystrophic

epidermolysis bullosa– Epidermolysis bullosa

simplex– Pachyonychia congenita

• siRNA targets mutant gene transcription products in these conditions and leads to decreased synthesis of aberrant proteins. From Pendaries et al, J Invest

Dermatol 132(6):1741-3

siRNA in Dermatology• siRNAs are also being studied as part of

melanoma treatments by targeting proteins critical to cell proliferation, preferentially affecting cancer cells.

From Zuckerman et al, J Invest Dermatol 131(2):453-60.

Limitations of siRNA

• Delivery of siRNAs into cells in vitro by transfection is a complex process with variable success.

• Translating siRNAs for therapeutic purposes into humans is complicated by factors of appropriate delivery to target cells and stimulation of host immune responses.

• Measuring and quantifying in vivo effects of therapeutic siRNA are challenging.