(A) The  introduction of the 1',2'-locked oxetane (1',3'-O-anhydro-beta-D-psicofurnosyl)thymine) modified nucleoside T

We have developed novel 1',2'-oxetane locked nucleosides, [1-(1',3'-O-anhydro-beta-D-psicofuranosyl) nucleosides] which have a unique fixed North-East sugar conformation. Historically we have started by synthesizing oxetane modified ribo-T. These North-East conformationally constrained  oxetane nucleoside, (T) had been incorporated as single, double, triple modifications into a 15 mer AON and targeted to a 15 mer RNA. Although there is a loss of Tm of ca 6oC  per modification, all the T modified AON/RNA hybrids were found to be substrate for RNase H as good as the native counter part  [PDF 313, 315, 329, 332]. CD failed to detect any structural perturbances of the modified hybrids compared to the native counterpart; however, the RNase H cleavage pattern clearly showed that the local conformational changes spanning a total of 5 nucleotides including the modification towards the 5'-end of AON (3'-end of RNA). This was evident from the fact that the 5 nucleotide region of the RNA strand, beginning from the nucleotide opposite to the T modification, became completely inactive to the catalytic cleavage reaction by RNase H owing to the local RNA/RNA type conformation. Since the site just after the 5 nucleotides were accessible for enzymatic cleavage, and the fact that the binding and cleavage sites are different for RNase H, it is evident that the structurally altered duplex region was suitable for enzyme binding but not for cleavage. By suitably placing the just three T modifications we have shown that the single cleavage site can be engineered in the 15mer AON/RNA hybrid duplex [PDF315].  This work clearly shows that owing to the Noth-type or North-East type conformational constrain introduced at the oligonucleotide level, leads to microenvironmental conformational alterations in the neighboring 4 nucleotides toward the 5'-end. This conformational transmission can be effectively mapped by RNase H, when CD and NMR fails to show any conformational heterogeinity in the local structure.





After the completion of our work  [PDF 314, 315, 329, 332] a similar study has been recently reported by Berlin Group for Wengels LNA modified AON/RNA duplexes (Nucleic Acids Res., 30, 1911(2002)). They  found that  RNase H can cleave of the gapmer/mixmer of LNA/RNA duplexes having 6 to 10 deoxynucleotide gaps. However, mixmer AONs with 3-5 deoxynucleotide gaps were found to be insensitive towards RNAse H promoted cleavage once hybridized to RNA . This shows that the conformational transmission of North-type sugar constrained LNA stretches upto 7 nucleotides to make it conform to RNA/RNA type structure, which is resistant to RNase H, whereas for oxetane modifications, we have shown it stretches upto 5 nucleotides. Quite expectedly, it was also found that the maximum cleavage efficiency was observed  for LNA gapmer as the size of the deoxynucleotide gap was increased, presumably because it adopted a substantial degree of DNA/RNA type structure .

Oxetane T-modified AONs can give at least 3 fold increment in endonuclease resistance [PDF 329]. We have also shown that the loss of caused by T modifications can be partially regained (+6 to +8o C) y the introduction of Dipyridophenazine (DPPZ) moiety at the 3' end of the oligochain which also made the AON stable towards 3'-exonuclease.

 To understand the substrate specificity (K ,  Vmax and kcat) of RNA cleavage by RNase H in oxetane modified AON/RNA hybrids, a detailed kinetic analysis of RNase H promoted cleavage of the RNA target in the triple T modified hybrid duplexes has been carried out [PDF 332]. It has emerged that the Vmax and the K were respectively ~2 and ~10 times more for the triple T oxetane modified AON/RNA duplex than those for the native counterpart, which shows the increase in the catalytic activity of the oxetane modified AON/RNA duplex almost 2 fold owing to the decreased affinity of the substrate toward the enzyme.

(B) Single and double oxetane C modifications [1-(1',3'-O-anhydro-ß-D-psicofuranosyl)cytidine] have also been evaluated [PDF 335], in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show ~3oC drop per modification in Tm of their hybrid AON–RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON–RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart.

A detailed Michaelis–Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON–RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the RNase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON–RNA duplexes.

All oxetane modified AON–RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, [Oxetane T and oxetane C modified mixmer AON with DPPZ at the 3'-end were found to be non-toxic from 1 µM to 20 µM in K562 Cell culture assays. A. P. Gewirtz, University of Pennsylvania, USA; personal communication.]).

Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the RNase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.

(C) Synthesis and enzymological studies of the oxetane-A and oxetane-G nucleos(t)ides

We have developed novel  synthesis of the oxetane-A and oxetane-G nucleosides [PDF 350 (G, A), 360] as well as their incorporations into antisense oligonucleotides (AONs), and comparison of their structural and antisense properties with those of the oxetane-T and -C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases). The oxetane-A and -G phosphoramidites were synthesized and incorporated into 20mer AONs and targeted to 20mer RNA, and their antisense properties have been evaluated in vitro.

The molecular structures of the oxetane-C, T, A, and G monomer units have been studied by means of the high-field 1H NMR and theoretical ab initio and MD simulations. The combined experimental and theoretical studies have demonstrated that the oxetane-fused furanose ring is indeed locked in the typical North-East-type conformation with the pseudo-rotational phase angle (P) and puckering  amplitude (fm) for the ab initio optimized geometries (6-31G* HF) varying from 39.8° < P < 42.8°, 35.1° < fm <36.6° for all four oxetane-modified nucleosides. The last 100 picoseconds (ps) of 0.5 nanosecond (ns) MD simulation starting from the respective ab initio geometries have shown for the oxetane-modified sugars accessible conformational range of P and fm to be 16° <P < 56°, 23 ° <fm <41°.

Oxetane-A and -G modified AONs have shown their target affinity with complementary RNA, and the thermodynamic parameters to be identical or very close to the native AON/ RNA duplex. However, the oxetane-C and oxetane-T modified duplexes showed large enthalpic destabilization and slight entropic stabilization compared to that of the native hybrid duplex. The large drop in enthalpy amounts to 32 to 37 kJ/mol drop in the free energy.

The global helical structure of all the oxetane modified AON/RNA hybrids, as revealed by the CD spectra, was found [PDF 350] very similar to the native AON/RNA duplex showing that the CD failed to detect the local conformational perturbations brought about by the North-East conformationally constrained oxetane modifications.

All of the oxetane-A and oxetane-G modified AON/RNA hybrid duplexes were found [PDF 350] to be good substrates for the E. coli RNase H1. In the oxetane-A and-G modified AON/RNA hybrids, except for one case, a region of 5 nucleotides in the RNA strand in the 3'-end direction from the site opposite to the oxetane modification, was found to be insensitive toward RNase H cleavage presumably owing to the local structural perturbations brought about by the conformationally constrained modifications.

Michaelis-Menten kinetic analysis showed that triple oxetane-A modified AON/RNA duplex has Vmax slightly lowerthan those of the native and the triple oxetane-C modified AON/RNA hybrids. However, the Km is very close to the native yielding a kcat/Km close to it. The triple oxetane-G modified AON/RNAduplex showed low Km and low Vmax compared to the nativecounterpart resulting in a kcat/Km value being half of that of the
native AON/RNA hybrid.

The conjugation of nontoxic DPPZ group at the 3'-end of the oxetane-A and -G modified AONs increases the Tm ofthe respective AON/RNA duplexes only by 1-2 °C in comparison with their unconjugated counterparts. However, the DPPZ conjugation has helped the AONs to achieve substantial stability against exonuclease (t1/2 more than 24 h) and nucleasesin human serum (t1/2 more than 9 h).

Modification of AONs with the oxetane-A and -G units failed to offer resistance toward DNase 1 endonuclease degradation in comparison with the oxetane-C modified AONs. Thus, DNase 1 appeared to have varying tolerance toward the oxetane-purine and oxetane-pyrimidine nucleotides.

This study  [PDF 350]  unravels the pros and cons of the AONs modified with the oxetane-purine and oxetane-pyrimidine nucleosides and provides valuable information regarding the optimal design of AONs having completely natural phospho-diester backbone for the therapeutic applications.

For further details see the original papers:
oxetane-T, -C, -A, -G [PDF 314, 315, 329, 332, 335 (C), 350 (G, A), 360]