RNA Chemistry

Overview of RNA Oligonucleotide Synthesis

The chemical synthesis of nucleic acids and related compounds play an essential role in numerous areas of the life sciences. For example, synthetic oligonucleotides are used as primers for polymerase chain reaction (PCR) (e.g. 20mer) and DNA sequencing studies, as well as for the creation of site-specific mutations. Certain artificial oligonucleotides have demonstrated usefulness as potential therapeutic reagents such as antisense molecules and also as probes in various biochemical studies. Recent progress in DNA microarray technology has required immobilization of pure synthetic oligonucleotides (50–60mer) on glass surfaces. In addition, based on the recently completed sequencing of the human genome, gene therapy and gene-based diagnosis have entered the realm of possibility, both of which require the use of natural and unnatural oligonucleotides of high purity.

The development of special RNA is one of the key discoveries in the field of RNA application. Short double-stranded RNAs (dsRNA), with a general chain length of 21-23mer and a two nucleotide 3’-end overhang, mediate specific gene suppression of mammalian cells. Such RNA is also called small interfering RNA (siRNA). siRNA guides endonucleolytic cleavage of the target RNA at a single site. The RNAi offers astronomical potentials for understanding and manipulating human diseases at the cellular level. Among the vast and rapidly exploding field of siRNA, some key developments taking place are summarized as:

n       Prediction of natural regulatory mechanism in the field of cell biology and functional genomics (this is expected to result in gene based drug discovery);

n       Gene control, gene knockdown, and target validation are currently being pursued with great vigor by vast number of researchers worldwide.

Internucleotide-bond formation is important for the synthesis of nucleic acids. Several methods have been developed for the syntheses of oligonucleotides, including the phosphodiester method, the phosphotriester method, the phosphate method, the phosphoramidite method, and the H-phosphonate method. An outline of these methods is provided in Fig. (1).Among the methods employed for internucleotide-bond formation, the phosphoramidite method is superior in many respects, including coupling efficiency, stability of building blocks, ease of automation, purity of the product, and synthetic applicability. For the synthesis of medium-size (longer than 20mer) oligonucleotides, the average yield of one-base elongation must exceed 99%. The amidite method meets this requirement. Phosphoramidites are a stable white powder and are easy to handle in a solid-phase synthesizer. The H-phosphonate method has also been applied for the synthesis of relatively long oligomers (more than 10mer); however, the purity of the final oligomers tends to be rather low.  

The synthesis cycle for RNA oligonucleotides consists of the same series of reactions as the cycle that is employed for Fast Deprotection DNA monomers. However, the rate of coupling for RNA monomers is slower, compared to that of DNA monomers (a coupling time of 10 minutes for RNA monomers is recommended compared to 90 seconds for DNA monomers). With the exception of the monomers and supports, RNA synthesis is accomplished with the same reagents as DNA synthesis. All RNA phosphoramidites from GenePharma Reagents are diluted with dry acetonitrile. Fast Deprotection CAP A is employed to prevent the transacylation of guanidite bases, similar to synthesis of tertbutylphenoxyacetyl (TAC) DNA monomers.

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Figure-1 RNA Synthesis Cycle