3'-deoxyadenosine (3-dA)(2'-5' linked) is deoxy at the 3-position of the ribose, instead of at the usual 2-position (note: the 3-deoxynucleotides of A, C, and T are also available from Gene Link). 3-deoxynucleotide (2,5-linked) modifications are used to substitute 2-5 phosphodiester linkages for the usual 3-5 phosphodiester linkages at some or all positions of an oligo. Oligonucleotides containing all, or primarily, 2,5-phosphodiester linkages selectively bind to complementary single-stranded 3,5-RNA over comparable 3,5-DNA (1,2). This property means that DNA oligos containing such linkages could be useful in either anti-sense applications or as ssRNA-specific probes.
Bhan et al. (2) studied the potential for 2,5-linked DNA oligos as anti-sense molecules. High selectivity for 3,5 RNA over 3,5 DNA was observed, presumably due to the 2,5-linkages destabilizing duplexes formed with 3,5 DNA more than those formed with 3,5-RNA (for 2,5 DNA:3,5 RNA duplexes, DeltaTm is only about 0.5 degC per 2,5 linkage substitution). Phosphorothiolation (which confers nuclease resistance) of 2-5 linkages lowers the Tm of 2,5 DNA:3,5 RNA duplexes even less, ~ 0.2 degC per phosphorothiolated 2,5-linkage substitution. (by contrast, phosphorothiolation of a 3,5 linkage lowers the Tm of 3,5 DNA:RNA duplexes by 0.5 to 2.0 degC). Thus, 2,5-linked DNA oligos show both high selectivity and good duplex stability for RNA target sequences. However, 2,5-linked DNA oligos, whether phosphorothiolated or not, do not support RNAse H activity when bound to complementary RNA. But, substitution of six or seven contiguous 3,5 phosphorothiolate linkages into a 2,5 phosphorothiolated oligo at an appropriate place (that is, making a 2,5/3,5 phosphorothiolated chimera restores the oligos ability to support RNAse H activity. Furthermore, 2,5-linked DNA oligos, whether phosphorothiolated or not, show little or no non-sequence specific binding to cellular proteins (by contrast, 3,5 DNA oligos show considerable levels of such binding.
In summary, this research suggests that 2,5/3,5 phosphorothiolated chimeric oligos, in which 6-7 of the linkages are 3,5 to ensure that it can support RNAse H activity, have considerable potential as anti-sense reagents, due to their high selectivity for complementary RNA targets, and minimal non-sequence specific binding to cellular proteins.
In 2004, Sinha and co-workers showed that 2,5-linked DNA has some capability to function as a template for polymerase-directed DNA synthesis of the complementary strand (3). The authors showed several polymerases, and HIV reverse transcriptase, can successfully use a string of 2-4 2,5-linked DNA nucleotides as a template to synthesize its complementary strand with high fidelity, and speculated that the polymerases were serving as a template for the template, i.e., compensating for structural deficiencies in the 2,5-linked DNA that, in non-enzymatic contexts, would preclude genetic information transfer for 2,5-linked DNA.
1. Giannaris, P.A.; Damha, M.J. Oligoribonucleotides containing 2,5-phosphodiester linkages exhibit binding selectivity for 3,5-RNA over 3,5-ssDNA. Nucleic Acids Res (1993), 21: 4742-4749.
2. Bhan, P.; Bhan, A.; Hong, M.K.; Hartwell, J.G.; Saunders, J.M.; Hoke, G.D. 2,5-linked oligo-3-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression. Nucleic Acids Res. (1997), 25: 40-41.
3. Sinha, S.; Kim, P.H.; Switzer, C. 2,5-Linked DNA Is a Template for Polymerase-Directed DNA Synthesis. J. Am. Chem. Soc. (2004), 126: 3310-3317.