RNA synthesis
The solid phase synthesis of RNA fragments is now a widespread technique in Molecular Biology. RNA oligonucleotides are becoming increasingly important in structural studies where they can be used as probes in Northern blotting or for in situ hybridization. In regulation, RNA oligonucleotides have been used in an antisense mode. The role of catalytic RNA (ribozymes and most importantly siRNA) as putative antisense therapeutics has sparked considerable interest in synthetic RNA.
Although on the whole it’s similar to DNA synthesis the additional 2’-OH reactive group of RNA introduces considerable complexity to the synthesis and requires an additional protective group. The use of the 2’-OH TBDMS protecting group has now become widely accepted as the best compromise for RNA synthesis of oligonucleotides up to 80 bases long.
siRNA
Custom RNAi Oligonucleotide Synthesis
RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Discovered in 1998, it has become clear now that RNA interference represents the major technological advance in Molecular Biology since the discovery of PCR and hence is called "The Next Big Thing in Biotech"
Mechanism of RNA interference (RNAi)
Upon introduction into the cell, long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference pathway. First the dsRNA’s are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC). The siRNA strands are then unwound to form activated RISCs. These activated RISCs then bind to complementary RNA molecules by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing.
Although there are different methods to generate siRNA for gene silencing, the easiest and most efficient way to achieve RNAi is to use synthetic small-interfering RNA (siRNA). siRNA are duplexes of short mixed oligonucleotides. These are typically made of 19 RNAs nucleotides followed by a 3’ dTdT overhang (Fig 1).
Fig 1: Principle of synthetic siRNA-mediated RNA interference
siRNA synthesis
For successful knockdown of your gene of interest, Biolegio provides you a powerful tool. Biolegio’s siRNA is synthesized with high quality chemicals. Synthesis is performed under stringent computer controlled conditions. Internal control functions measure the base coupling efficiency and guarantee the siRNA oligo to be of the highest quality standard, as you can expect from all products of Biolegio.
All siRNAs contain 19 RNA + 2 DNA bases overhang. Any mixture of DNA and RNA bases is possible, however by default dTdT overhangs are added to the 3’ ends. This typically provides a more reliable synthesis than UU as well as helps to protect the RNA from degradation. For RNA overhang, please contact us. Optimized deprotection and desalting procedures are handled to i9nsure the quality of your product. In order to ensure maximum stability, all siRNAs are provided lyophilized in individual tubes.
siRNA is standard deprotected, desalted and ready to use. Furthermore, PAGE purification is available upon request to generate a purity of > 97%.
Synthesis scales:
200 nmol synthesis scale, 45 nmol yield of a 21 mer
1000 nmol synthesis scale, 90 nmol yield of a 21 mer
siRNA’s are delivered single-stranded, and can be easily annealed.
Care and Handling of siRNA oligos
General Handling Instructions
RNA oligos are susceptible to degradation by exogenous ribonucleases introduced during handling. RNase-free reagents and supplies should be used.
Resuspension of Single-Stranded RNA Oligos
Oligonucleotides may be re-suspended at a convenient concentration in RNase-free sterile water The use of DEPC-treated water is not recommended. Store at -20°C.
Dried RNA oligos are stable for 1 year at -20°C. Once re-suspended, oligonucleotides solutions are best kept frozen at -20 °C for several weeks and may remain stable for several months. The most important factor in storing working solutions is using nuclease-free, sterile water. Drying down of your oligos and keeping them at -20 °C is recommended for long-term storage.
Annealing of RNA Oligos
Combine the sense and antisense RNA oligonucleotides, water, and 5X RNA Annealing Buffer in a sterile vial. The final concentration should be 20 - 100uM for each oligo in 1X Annealing Buffer. Heat for 1 min at 90C, followed by incubation for 1 hr at 37°C. Once annealed, the double stranded RNA is more nuclease resistant and ready to transfect. It should be stored at -20°C.
1 x Annealing Buffer:
100mM Potassium Acetate
30mM HEPES-KOH at pH 7.4
2mM Magnesium Acetate
Store at -20°C