RNA Oligo Modifications Applications

RNA Oligo Modifications Applications

Gene Link specializes in the synthesis of complex RNA modified oligos, RNA fluorescent probes with quenchers, chimeric oligos containing various combinations of DNA, 2'O methyl bases, 2'F bases and phosphorothioate linkages.

RNA and 2'-O-methyl RNA oligonucleotide synthesis is performed at Gene Link using the b -cyanoethyl chemistry and state of the art synthesizers. These involve proprietary software protocols with long coupling times and specialized cycles to obtain ultra clean oligos.
RNA oligos are susceptible to degradation to the same extent as native RNA extracted from various sources. An attractive alternate to prevent degradation from nucleases is the use of 2'F and 2'-O- methyl RNA bases, when specific 2'OH is not required. The 2'-O- methyl oligonucleotides confer considerable nuclease resistance and are similar in hydrogen bonding properties to RNA/RNA than the lower RNA/DNA binding property. The coupling efficiency of 2'-O- methyl phosphoramidite is also higher than the RNA monomers resulting in higher yield of full length oligos.
Gene Link also offers custom synthesis of RNA and DNA chimeric oligos with investigator specified ribo or deoxy bases or 2'-O-methyl bases. The chimeric oligos can also be synthesized with the regular phoshodiester bonds or substituted with phosphorothioate linkages. The combination of 2'-O- methyl RNA bases with phosphorothioate internucleotide linkages imparts these oligos greater nuclease resistance which is particularly useful for antisense studies (please refer to our technical sheet on Antisense Oligonucleotides for other modifications). Custom phosphorothioate oligonucleotides synthesized by Gene Link can be specified to have all the diester bonds substituted or only some selected diester linkages depending upon the researchers experimental requirement. Substitution of all diester linkage is recommended to provide greater nuclease resistance.

2'-Fluoro-deoxy bases are classified as a 2'-Fluoro RNA monomer. The substitution of a highly electronegative fluorine atom for the 2'-OH group of the ribose results in the ring of a 2'-F-ribonucleoside having a C3'-endo (i.e., RNA-type) conformation. Consequently, an RNA oligonucleotide containing a 2'-F RNA monomer adopts the more thermodynamically stable A-form helix on hybridization to a target (1).

2'-F RNA modifications have been shown to be particularly useful in the following oligo-based applications:

(a) Anti-sense DNA oligos: When 2'-F RNA is incorporated into an anti-sense DNA oligo (resulting in a 2'-F RNA/DNA chimeric), the Tm of its duplex with RNA increases relative to that formed by an unmodified anti-sense DNA oligo by 1.8°C per 2'-F RNA residue added (1). The DNA/RNA duplex formed between a chimeric 2'-F-RNA/DNA anti-sense oligo and its RNA target also is a substrate for RNase H. With respect to nuclease resistance, while 2'-F RNA nucleotides do provide some nuclease resistance when incorporated into DNA, phosphorothiolation of the 2'-F RNA phosphodiester linkages is recommended, because it strongly enhances such resistance. This effect becomes particularly important if the 2'-F RNA nucleotide is to be incorporated at one or more of the first three positions of the 5'- or 3'-ends of the oligo.

(b) Aptamers: The addition of 2'-F pyrimidines (C, U) into RNA aptamers has been shown to provide them with both considerably increased nuclease resistance and equal or higher binding affinity for their ligands, compared to the corresponding unmodified aptamers (2,3). The incorporation of 2'-F RNA nucleotides into RNA aptamers as an optimization strategy is becoming an increasingly common practice.

(c) siRNA: siRNA synthesized with 2'-F pyrimidines have been shown to have greatly increased stability in plasma compared to 2'-OH siRNA (4,5). In one study, levels of inhibition for 2'-F siRNA and 2'-OH siRNA, in cell culture and in vivo using BALB/c mice transfected with pGL3 luciferase, were similar over time (4). In another study, siRNA fully substituted with both 2'-F RNA and 2'-O-Methyl RNA nucleotides displayed not only enhanced stability in plasma, but also >500-fold increase in capability to down-regulate gene expression compared with the corresponding unmodified siRNA (5).

(d) LNA Alternative: The increased thermal stability and nuclease resistance provided by 2'-F RNA residues make them attractive as lower-cost substitutes in any application involving LNA-modified oligos.

References

REFERENCES:
1. Kawasaki, A.M., et al., Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease resistant antisense compounds with high affinity and specificity for RNA targets, Journal of Medicinal Chemistry (1993), 36: 831-841.
2. Khati, M.; Schuman, M.; Ibrahim, J.; Sattentau, Q.; Gordon, S.; James, W. Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2'F-RNA aptamers. J Virol, (2003). 77: 2692-8.
3. Goringer, H.U.; Adler, Annette; Forster, Nicole; Homann, Matthias. Post-SELEX Chemical Optimization of a Trypanosome-Specific RNA Aptamer. Combinatorial Chemistry & High Throughput Screening (2008), 11: 16-23.
4. Layzer, J.M.; McCaffrey, A.P.; Tanner, A.K.; Huang, Z.; Kay, M.A.; Sullenger, B.A. In vivo activity of nuclease-resistant siRNAs. RNA (2004), 10, 766-71.
5. Allerson, C.R.; et al. Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. Journal of Medicinal Chemistry (2005), 48: 901-904.