RNase 2 -modified oligonucleotides

Inoue, H., et al. (1987). Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 215, 327—330. 

A series of ODNs containing a mixture of a- and p-anomeric nucleotides as well as 5, 5 or 3, 3 polarity reversal gapmers were targeted to the erbB-2 oncogene to examine RNase H activity. It was found that a p-anomeric window of three nucleotides was sufficient to allow RNase H activity despite the reversal of polarity within the ODN. 2 -0-modified oligonucleotides containing an inverted 3 -3 thymidine at the terminus have been shown to lead to higher duplex stabilities and resistance to nucleases. ... 

Arabino- and 2 -arabino-fluoro nucleic acids (ANA and 2 -F-ANA, respectively) have been demonstrated to form hybrids with RNA that are capable of activating RNase H. The properties and potential applications of such modified oligonucleotides have been reviewed. The effect of substituting arabinonucleo-sides into the DNA priming site of the HIV-1 RT has been examined. The introduction of AraC at the 3 -end of the primer resulted in stalling after the addition of four nucleotides, whereas dC or riboC had no effect. The effect was observed for arabinonucleotides at the first five positions of the primer. 

Modified Oligonucleotides with Ability to Activate RNase H, 152... 

Modified Oligonucleotides with Ability to Activate RNase H. Uniformly 2 -modified oligonucleotides are not capable of activating RNase H in an oligonucleotide/RNA hybrid. However, recent reports show that hybrids of RNA and arabinonucleic acids [the 2 -stereoisomer of RNA based on o-arabinose instead of the natural o-ribose (2 -arabino-OH, ANA) and 2 -deoxy-2 -fluoro-D-arabinonucleic acid (2 F-ANA)] are substrates of RNase H (358,359) (Fig. 5.9). However, ANA/RNA hybrids had a lower value, compared to that of the control DNA/RNA hybrids. This destabilization (AT, = -1.0°C/modification) is presumed to derive from steric interference by the -C2 -OH group, which is oriented into the... 

The length and specific design, however, may vary depending on the intended use and on how important the affinity, nuclease resistance, and the RNase H/RISC recmitment (see below) is for the proposed application. In the classic designs described above, the total length of LNA oligonucleotides is 16 mers, and the oligonucleotides are fully PS-modified. 

Target RNA species that have been reported to be inhibited include HIV (28), vesicular stomatitis virus (VSV) (76), n-myc(109), and a number of normal cellular genes (110-113). However, to demonstrate that RNase H is not involved in effects observed, it is necessary to use antisense drugs that do not form duplexes that are RNase H substrates (e.g., fully modified 2 -oligonucleotides). 

Figure 39. Left Required assembly for trans-active RNA cleavage by RNase P-like Ml RNA protein complex in E. coli. Right Assembly of two oligonucleotides forming a hammerhead-like ribozyme (N variant bases). Modified from [342].

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