Reverse RNA Amidites and Supports

Reverse RNA Amidites and Supports:

• ChemGenes has introduced reverse RNA technology for synthesis of very pure therapeutic grade natural RNA in reverse 5→3′ direction (Figure 1).
•  The novel reverse RNA amidites, A(N-Bz), C(N-Bz), C(N-Ac), G(iPrPAC) and U are produced with HPLC purity of  >98% and ³¹P NMR purity of >99%.

Figure 1: Schematic illustration of synthesis of RNA using reverse RNA technology.

Salient Features:

• Shorter coupling time (~2 min) is used.
• Higher coupling efficiencies are observed (Comparable to standard DNA amidites).
• Long chain RNA oligonucleotides are conveniently synthesized.
• No need for process change/Re-validation (Same 2′-TBDMS protection as conventional RNA amidites).
• High purity of crude & purified oligomers produced by this technology.
• Ultra high purity 3′-modifications synthesized with ease.
• No 3′-isomer detected (Isomer formation does not occur during oligo synthesis and purification process).
• Complete absence of N-1 impurities - RNAs synthesized by reverse RNA synthesis are much pure than that of synthesized by conventional 3′→5′-direction.
• Macromolecules attached RNA (such as cholesterol & PEG) are easy to purify after reverse direction RNA synthesis (see Figure 2 & 3 purified 3′-conjugates).

• N+1 impurities are essentially absent in RNAs synthesized by reverse RNA synthesis method. It is postulated that in reverse RNA amidites, the 3′-DMTr group is not cleaved by 5-ethylthiotetrazole (ETT) during the coupling step of oligonucleotide synthesis.

Applications:

1. Synthesis of high purity therapeutic grade RNA and siRNA.

2. With our proprietary technology, we have successfully synthesized long RNA oli­gonucleotides up to 200 bases with high purity & efficiency. Figure 4 and 5 shows ESI MS and Gel analysis of 74-mer long RNA that is synthesized by reverse RNA technology.

Figure 4: ESI MS analysis of 74-mer RNA synthesized using reverse amidites (scale 10.0 µm).

Figure 5: Gel analysis of 74-mer RNA synthesized by reverse RNA amidites.

3. Attachment of reporter groups such as cholesterol, long chain aliphatic chains, fluorophores, triethylene glycols, hexaethylene glycols and PEG at the 3′-end of the RNA can be achieved easily by direct coupling with these amidites.

4. Attachment of Polyethylene Glycols such as PEG 2000 and PEG 4500 amidites at the 3′-end of the RNA molecule.

5. For easy attachment of 3′-thiol modification. 3′-Disulfides from readily available amidites, via C-3 disulfide, C-6 disulfide.

6. 3′-Biotin attachment via biotin amidite in a single step and avoiding biotin CPG.

7. Modification of 3′-end of the sense strand of siRNA. The modification of the overhang of the sense strand (3′-End) of siRNA is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition. Useful modification for improvement of delivery of siRNA can be easily designed.

References:

1. Srivastava, S. C.; Srivastava, N. P.; RNA Synthesis in the Reverse Direction, 2011, PCT Int. App., WO2011103468.
2. Srivastava, S. C.; Srivastava, N. P.; RNA Synthesis in the Reverse Direction, 2011, PCT App., Pub. US20110137010.
3. Srivastava, S. C.; Pandey, D.; Srivastava, N.; Bajpai, S. P. RNA Synthesis in the Reverse Direction, 2010, WO2010027512.
4. Srivastava, S. C.; Pandey, D.; Srivastava, N.; Bajpai, S. P. RNA Synthesis in the Reverse Direction, 2011, Curr. Pro. in Nuc. Acid Chem.; Unit 3.20.