Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

Oligoribonucleotide analogues containing a mixed backbone of phosphodiester and formacetal internucleoside linkages, together with vicinal 2'-O-methyl groups.

Oligoribonucleotides containing formacetal internucleoside linkages have been prepared and studied by UV melting experiments. In RNA duplexes, the formacetal substitution is stabilizing (Deltat(m)=0 to +0.9 degrees C per modification) at physiological salt concentrations (0.1 M) but destabilizing (Deltat(m)=-0.4 to -0.8 degrees C per modification) at high salt concentrations (1 M); this suggests that reduction of electrostatic repulsion contributes substantially to the stabilization. The presence of 2'-O-Me substituents increases the stabilities of the duplexes (Deltat(m)=+0.5 to +1.1 degrees C per modification). The positive effects of formacetals and 2'-O-Me groups were independent and additive. (1)H NMR studies on monomeric model compounds containing 3'-(ethyl phosphate) or 3'-O-ethoxymethyl groups showed that the formacetal and 2'-O-Me substitutions shift the conformational equilibria of the ribose residues towards the North conformers by 5 to 12 %. Although the preference for the North conformers qualitatively correlates with increased duplex stabilities, changes in thermodynamic parameters (DeltaH degrees and TDeltaS degrees ) for formation of oligonucleotide duplexes and differences in dependence on concentrations of sodium acetate, sodium chloride and sodium perchlorate suggest that solvation effects are also important for the duplex stabilities. Overall the formacetal linkages fit well in A-type RNA duplexes, making them potentially interesting modifications for RNA-based gene-control strategies (e.g., antisense and RNA interference).

Toward amide-modified RNA: synthesis of 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides.

Recent discovery of RNA interference has reinvigorated the interest in chemically modified RNA. Chemical approaches may be used to optimize properties of small interfering RNAs, such as thermal stability, cellular delivery, in vivo half-life, and pharmacokinetics. From this perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly interesting modifications that so far have not been tested in RNA interference. Amides are remarkably good mimics of the phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide coupling chemistry. The synthetic challenge that has hampered the progress in this field has been preparation of monomeric building blocks for such couplings, the nucleoside amino acid equivalents. Herein, we report two synthetic routes to enantiomerically pure 3'-aminomethyl-5'-carboxy-3',5'-dideoxy nucleosides, monomers for preparation of amide-modified RNA. Modification of uridine, a representative of natural nucleosides, using nitroaldol chemistry gives the target amino acid in 16 steps and 9% overall yield. The alternative synthesis starting from glucose is somewhat less efficient (17 steps and 6% yield of 3'-azidomethyl-5'-carboxy-3',5'-dideoxy uridine), but provides easier access to modified nucleosides having other heterocyclic bases. The syntheses developed herein will allow preparation of amide-modified RNA analogues and exploration of their potential as tools and probes for RNA interference, fundamental biochemistry, and bio- and nanotechnology.

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions.

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.