2'-Fluoro Uridine 3'-lcaa CPG 500Å

As RNA gets rapidly degraded to the nucleases, it serves unsuitable for nucleic acid based applications. Hence, various 2′-RNA modified monomers such as 2′-O-alkyl and 2′-fluoro have been synthesized to achieve suitable candidate for nucleic acid based therapeutics and diagnostics. 2′-Fluoro modification found to be one of the very promising candidates among other 2′-modifications. In 2′-fluoro oligonucleotides, absence of 2′-hydroxyl group makes the inter-nucleotide phosphate linkages are more stable to a chemical hydrolysis at a high pH compared to that of in unmodified RNA oligonucleotides. The increased chemical stability of 2′-deoxy-2′-fluoro RNA oligonucleotide analogs is accompanied by high resistance to ribonucleases and, consequently, leads to a longer survival time of 2′-fluro RNAs in biological environment.

Similar to the hydroxyl group at 2′-position, 2′- fluoro also preorganizes the sugar ring in a C3′-endo conformation. Therefore, 2′-fluoro oligonucleotides adopt an A-form helix on hybridization with a target strand. 2′-Fluoro RNA oligonucleotides can often be purified using the same method as standard DNA oligonucleotides. Oligonucleotides with modified with 2′-fluoro monomers have been found to increase the thermal stabilities upon hybridization to target RNA (1-2 ^ºC per modification) and DNA (1.3 ºC per modification) compare to natural RNA:RNA and DNA: DNA duplexes, respectively. 2′-Fluoro is a poor hydrogen-bond acceptor in the minor groove, whereas 2′-OH is extensively hydrated and serves as a bridge head for water molecules linking strands across the minor groove.¹ Thermodynamic data of 2′-fluoro oligonucleotides indicated that the higher stability is entirely based on favorable enthalpy and not entropy.²

Oligonucleotides containing 2′-deoxy-2′-fluoro ribonucleotide have found numerous beneficial applications in ribozyme,³ antisense,⁴siRNA,⁵ miRNA,⁶and aptamer-based⁷ nucleic acid therapeutics. Moreover, antisense oligonucleotides bearing 2′-fluoro ribonucleotides were recently found to exhibit favorable properties for modulating splicing, relative to other 2′-modifications.⁸ 2′-Deoxy-2′-fluoro nucleosides have been found to display significant biological activity as nucleosides and as triphosphates, and are used as versatile probes for DNA and RNA polymerases.⁹ siRNAs with extensively modified with 2′-fluoro pyrimidines showed increased nuclease stability, reduced immune stimulation, and in some cases exhibited favorable activity in vitro and in vivo, relative to unmodified control RNA.¹⁰ However, duplexes of 2′-fluoro oligonucleotides and RNA do not support RNAse H activity. Aptamers composed of 2′F-RNA bind targets with higher affinities and are more resistant to nucleases, compared to RNA aptamers.

Because of interesting properties of 2′-fluoro-2′-deoxy modifications there is great demand for 2′-fluoro CE phosphoramidites and it’s supports. ChemeGenes offers all four (A, C, G and U) phoshoramidites and supports of 2′-fluro modifications. We hope that, full set of these phosphoramidites and supports from us will serve as a very useful research tools for our customers in the field of 2′-fluro oligonucleotides.

Deprotection and Purification Protocol:

Step 1: Cyanoethyl group removal either with a) 10% diethylam MeCN¹¹ (v/v) for 3 min at rt or with b) 50% triethylamine in acetonitrile (v/v) for 30 min at rt.

Step 2: Base deprotection with conc. aq. NH₄OH (30%) for 16 hr at 37 °C

Step 3: Purification:

a) Cartridge purification:

Purification of the crude 2′F-RNA oligo can be achieved using ChemGenes puri-pak purification cartridge (ChemGenes Cat # CSS-3920). The ChemGenes purification columns are designed for efficient and fast desalting of oligonucleotides. They are recommended for single use, and 50 OD units (obtained from 0.2 µm scale oligo synthesis) can be loaded

b) HPLC purification

If the 2′F-RNA obtained after cartridge purification is not sufficiently pure (depending on the purity requirements) is subsequently purified by HPLC.

References:

1) Egli, M. et. al. Biochemistry 1996, 35, 8489.

2) Pallan, P. S. et. al. Nucleic Acids Res. 2011, 39, 3482.

3) Pieken, W. A. Science 1991, 253, 314.

4) Kawasaki, A. M. et. al. J. Med. Chem. 1993, 36, 831.

5) Allerson, C. R. et. al. J. Med. Chem. 2005, 48, 901.

6) Davis, S. et. al. Nucleic Acids Res. 2009, 37, 70.

7) Ng, E.W. M. Nat. Rev. Drug Discovery 2006, 5, 123

8) Rigo, F. et. al. Nat. Chem. Biol. 2012, 8, 555.

9) Viani, F., V.A. Ed. Chapter 13, pp 419-449, John Wiley & Sons Ltd. 1999; Wright G.E. et. al. Ther. Pharmacol. 1990, 47, 447.

10) Manoharan, M. et. al. Angew. Chem. Int. Ed. 2011, 50, 2284.

11) Bhan A. Et al. US Patent 6,887,990.

Oligonucleotide Synthesis Reagents

Mononucleotides & Nucleosides

Other Reagents

2'-Fluoro & 2'-Amino Amidites and Supports

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