Five- and six-membered conformationally locked 2',4'-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.     

Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability,and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases

Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, 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 approximately 3 degrees C drop per modification in melting temperature (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 R Nase 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, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase 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.     

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.     

Gapmer Antisense Oligonucleotides Containing 2′,3′-Dideoxy-2′-fluoro-3′-C-hydroxymethyl-β-d-lyxofuranosyl Nucleotides Display Site-Specific RNase H Cleavage and Induce Gene Silencing

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2′-deoxy-2′-fluoro-3′-C-hydroxymethyl-β-d -lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2′-fluoro-3′-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.     

Carba-LNA-5MeC/A/G/T modified oligos show nucleobase-specific modulation of 3'-exonuclease activity, thermodynamic stability, RNA selectivity, and RNase H elicitation: synthesis and biochemistry

Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonucleotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc.2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 °C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl ((Me)C), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (ΔT(m) = 2.9 °C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, ΔT(m) = 2.2 °C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-(Me)C with trace of cENA-(Me)C (cLNA-(Me)C, ΔT(m) = 1.8 °C/modification) and 7'-R-Me-cLNA-A (cLNA-A, ΔT(m) = 0.9 °C/modification). cLNA-(Me)C modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -(Me)C modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a specific manner, which is important for the design of oligos for therapeutic purposes. The cLNA-(Me)C modified AON/RNA, however, has been found to be degraded 4 times faster than cLNA-A and G modified counterparts. By appropriately choosing the carba-LNA modification sites in AON strands, the digestion of AON:RNA can be either totally repressed or be limited to cleavage at specific sites or at a single site only (similar to that of catalytic RNAzyme or DNAzyme). Considering all physico- and biochemical aspects of cLNA modified oligos, the work suggests that the cLNA modified antisense oligos have the potential of being a promising therapeutic candidate due to their (i) higher nucleobase-specific RNA affinity and RNA selectivity, (ii) greatly improved nuclease stability, and (iii) efficient RNase H recruitment capability, which can induce target RNA cleavage in a very specific manner at multiple or at a single site, in a designed manner.     

Synthesis of 2',4'-propylene-bridged (carba-ENA) thymidine and its analogues: the engineering of electrostatic and steric effects at the bottom of the minor groove for nuclease and thermodynamic stabilities and elicitation of RNase H

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH(2)/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C═N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance but 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH(2) substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.     

Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues

We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the 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).     

Chemical and structural implications of 1',2'- versus 2',4'- conformational constraints in the sugar moiety of modified thymine nucleosides

In order to understand how the chemical nature of the conformational constraint of the sugar moiety in ON/RNA(DNA) dictates the duplex structure and reactivity, we have determined molecular structures and dynamics of the conformationally constrained 1',2'-azetidine- and 1',2'-oxetane-fused thymidines, as well as their 2',4'-fused thymine (T) counterparts such as LNA-T, 2'-amino LNA-T, ENA-T, and aza-ENA-T by NMR, ab initio (HF/6-31G** and B3LYP/6-31++G**), and molecular dynamics simulations (2 ns in the explicit aqueous medium). It has been found that, depending upon whether the modification leads to a bicyclic 1',2'-fused or a tricyclic 2',4'-fused system, they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and the backbone torsions as well as by characteristic North-East type sugar conformation (P = 37 degrees +/- 27 degrees , phi(m) = 25 degrees +/- 18 degrees ) of the 1',2'-fused systems, and (ii) pure North type (P = 19 degrees +/- 8 degrees , phi(m) = 48 degrees +/- 4 degrees ) for the 2',4'-fused nucleosides. Each group has different conformational hyperspace accessible, despite the overall similarity of the North-type conformational constraints imposed by the 1',2'- or 2',4'-linked modification. The comparison of pK(a)s of the 1-thyminyl aglycon as well as that of endocyclic sugar-nitrogen obtained by theoretical and experimental measurements showed that the nature of the sugar conformational constraints steer the physicochemical property (pK(a)) of the constituent 1-thyminyl moiety, which in turn can play a part in tuning the strength of hydrogen bonding in the basepairing.     

Structural rationalization of a large difference in RNA affinity despite a small difference in chemistry between two 2'-O-modified nucleic acid analogues

Chemical modification of nucleic acids at the 2'-position of ribose has generated antisense oligonucleotides (AONs) with a range of desirable properties. Electron-withdrawing substituents such as 2'-O-[2-(methoxy)ethyl] (MOE) confer enhanced RNA affinity relative to that of DNA by conformationally preorganizing an AON for pairing with the RNA target and by improving backbone hydration. 2'-Substitution of the ribose has also been shown to increase nuclease resistance and cellular uptake via changes in lipophilicity. Interestingly, incorporation of either 2'-O-[2-(methylamino)-2-oxoethyl]- (NMA) or 2'-O-(N-methylcarbamate)-modified (NMC) residues into AONs has divergent effects on RNA affinity. Incorporation of 2'-O-NMA-T considerably improves RNA affinity while incorporation of 2'-O-NMC-T drastically reduces RNA affinity. Crystal structures at high resolution of A-form DNA duplexes containing either 2'-O-NMA-T or 2'-O-NMC-T shed light on the structural origins of the surprisingly large difference in stability given the relatively minor difference in chemistry between NMA and NMC. NMA substituents adopt an extended conformation and use either their carbonyl oxygen or amino nitrogen to trap water molecules between phosphate group and sugar. The conformational properties of NMA and the observed hydration patterns are reminiscent of those found in the structures of 2'-O-MOE-modified RNA. Conversely, the carbonyl oxygen of NMC and O2 of T are in close contact, providing evidence that an unfavorable electrostatic interaction and the absence of a stable water structure are the main reasons for the loss in thermodynamic stability as a result of incorporation of 2'-O-NMC-modified residues.     

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.     

Structure and nuclease resistance of 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNAs

Combining the structural elements of the second generation 2'-O-methoxyethyl (MOE) and locked nucleic acid (LNA) antisense oligonucleotide (AON) modifications yielded the highly nuclease resistant 2',4'-constrained MOE and ethyl bicyclic nucleic acids (cMOE and cEt BNA, respectively). Crystal structures of DNAs with cMOE or cEt BNA residues reveal their conformational preferences. Comparisons with MOE and LNA structures allow insights into their favourable properties for AON applications.     

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Solution structure of a HNA-RNA hybrid

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.     

A novel RNA motif based on the structure of unusually stable 2',5'-linked r(UUCG) loops

We have recently shown that hairpins containing 2',5'-linked RNA loops exhibit superior thermodynamic stability compared to native hairpins comprised of 3',5'-RNA loops [Hannoush, R. N.; Damha, M. J. J. Am. Chem. Soc. 2001, 123, 12368-12374]. A remarkable feature of the 2',5'-r(UUCG) tetraloop is that, unlike the corresponding 3',5'-linked tetraloop, its stability is virtually independent of the hairpin stem composition. Here, we determine the solution structure of unusually stable hairpins of the sequence 5'-G(1)G(2)A(3)C(4)-(U(5)U(6)C(7)G(8))-G(9)(U/T(10))C(11)C(12)-3' containing a 2',5'-linked RNA (UUCG) loop and either an RNA or a DNA stem. The 2',5'-linked RNA loop adopts a new fold that is completely different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a). U5.G8 wobble base pairing, with both nucleotide residues in the anti-conformation, (b). extensive base stacking, and (c). sugar-base and sugar-sugar contacts, all of which contribute to the extra stability of this hairpin structure. The U5:G8 base pair stacks on top of the C4:G9 loop-closing base pair and thus appears as a continuation of the stem. The loop uracil U6 base stacks above U5 base, while the cytosine C7 base protrudes out into the solvent and does not participate in any of the stabilizing interactions. The different sugar pucker and intrinsic bonding interactions within the 2',5'-linked ribonucleotides help explain the unusual stability and conformational properties displayed by 2',5'-RNA tetraloops. These findings are relevant for the design of more effective RNA-based aptamers, ribozymes, and antisense agents and identify the 2',5'-RNA loop as a novel structural motif.     

Synthesis of a novel bicyclic nucleoside restricted to an S-type conformation and initial evaluation of its hybridization properties when incorporated into oligodeoxynucleotides.

The phosphoramidite (1S,3R,4S)-3-(2-cyanoethoxy(diisopropylamino)phosphinoxymethyl)-5-N-(4-monomethoxytrityl)-1-(uracil-1-yl)-5-aza-2-oxabicyclo[2.2.1]heptane 18 of a novel bicyclic nucleoside structure was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 3. Conformational analysis of its structure verified its expected S-type furanose conformation, and the secondary amino group in the 4'-position allowed for incorporation into oligonucleotides using 5' --> 3' directed oligonucleotide synthesis as previously described for phosphoramidates. Thermal denaturation studies showed rather large decreases in duplex stabilities of -4.3 and -2.7 degrees C per modification toward complementary DNA and RNA, respectively.     

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.     

Design, synthesis, and properties of 2',4'-BNA(NC): a bridged nucleic acid analogue

The novel bridged nucleic-acid analogue 2',4'-BNA(NC) (2'-O,4'-C-aminomethylene bridged nucleic acid), containing a six-membered bridged structure with an N-O linkage, was designed and synthesized efficiently, demonstrating a one-pot intramolecular NC bond-forming key reaction to construct a perhydro-1,2-oxazine ring (11 and 12). Three monomers of 2',4'-BNA(NC) (2',4'-BNA(NC)[NH], [NMe], and [NBn]) were synthesized and incorporated into oligonucleotides, and their properties were investigated and compared with those of 2',4'-BNA (LNA)-modified oligonucleotides. Compared to 2',4'-BNA (LNA)-modified oligonucleotides, 2',4'-BNA(NC) congeners were found to possess: (i) equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power, (ii) much better RNA selective binding, (iii) stronger and more sequence selective triplex-forming characters, and (iv) immensely higher nuclease resistance, even higher than the S(p)-phosphorthioate analogue. 2',4'-BNA(NC)-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.     

Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.