Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Syntheses of alternating oligo-2'-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, whose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, contain 2'-O-methylribonucleosides and alternating methylphosphonate and phosphodiester internucleotide linkages. The methylphosphonate and phosphodiester linkages of these oligomers are highly resistant to hydrolysis by exonuclease activity found in mammalian serum and to endonucleases, such as S1 nuclease. The oligomers were prepared using automated phosphoramidite chemistry and terminate with a 5'-phosphate group, which provides an affinity handle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementary RNA 18-mer, mini-TAR RNA. The T(m) of this duplex is 71 degrees C, which is similar to that of the duplex formed by the corresponding all phosphodiester 15-mer. Introduction of two mismatched bases reduces the T(m) by 17 degrees C. The apparent dissociation constant, K(d), for the 1676/mini-TAR RNA duplex as determined by an electrophoretic mobility shift assay at 37 degrees C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K(d) = 20 nM), whereas no binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K(d) that is similar to that of 1676, a result that suggests only part of the oligomer binds. When two of the methylphosphonate linkages in the region complementary to the 6-base loop are replaced with phosphodiester linkages, the K(d) is reduced by approximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loop, and that these interactions are sensitive to presence and possibly the chirality of the methylphosphonate linkages in the oligomer. The high affinities of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis suggests their potential utility as antisense agents in cell culture.     

Selective modification of cytosines in oligodeoxyribonucleotides.

A single deoxycytidine residing in an oligodeoxyribonucleotide which also contains 5-methyldeoxycytidines can be selectively derivatized with various alkylamines by sodium bisulfite-catalyzed transamination. Selective transamination results because 5-methylcytosine, unlike cytosine, does not form a bisulfite adduct. When the reaction is carried out at pH 7.1, transamination in the oligomer appears to occur to greater than 95% with little or no deamination. This procedure has been used to introduce aminoalkyl or carboxyalkyl side chains at the N4-position of a deoxycytidine in oligonucleotides. These side chains contain potentially reactive amine or carboxy groups which could serve as a sites for further conjugation of the oligomer with a variety functional groups. Oligonucleotides which carry these side chain form duplexes and triplexes with appropriate complementary single-stranded or double-stranded oligodeoxyribonucleotide target molecules. The stabilities of the duplexes are similar to those formed by unmodified oligomers, whereas the stability of the triplexes is approximately 18 degrees C lower than that formed by unmodified oligomers.     

Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ionic strength on the hybridization of oligodeoxynucleotides with reduced charge due to methylphosphonate linkages to unmodified oligodeoxynucleotides containing the complementary sequence

A 12-mer oligodeoxynucleotide containing 10 methylphosphonate bonds and 1 phosphodiester bond was shown to bind specifically to the restriction endonuclease fragment containing complementary DNA in a Southern blot. This 12-mer as well as 14-mer oligodeoxynucleotides containing 3 methylphosphonate and 10 phosphodiester bonds was used to examine the effect of reduced charge on the thermodynamics of binding to complementary DNA or complementary oligodeoxynucleotides with additional nucleotides overlapping both the 3' and 5' ends. The 14-mer oligodeoxynucleotides were synthesized with one methylphosphonamidite (A, C, G, or T). Melting profiles were examined by spectrophotometry for the 14-mers and by a gel-shift assay for the 12-mer. Nearest-neighbor free energy values were compiled for predicting concentration-dependent melting temperatures for all oligodeoxynucleotide hybridizations, including those involving adjacent dG residues. The free energy contribution to duplex formation from the dangling ends was about 1 kcal/mol. The free energy decrement due to introduction of each methylphosphonate linkage was -0.75 kcal/mol in high salt independent of the methylphosphonamidite used for synthesis of the oligodeoxynucleotide. However, the change in charge per nearest-neighbor base pair decreased from 0.26 to 0.0 when the nearest-neighbor base pair contained one methylphosphonate. Thus at very low salt, methylphosphonate-substituted oligodeoxynucleotides form more stable hybrids than analogous phosphodiester sequences. The 12-mer with 10 methylphosphonate bonds outcompetes the analogous phosphodiester 12-mer below 0.01 M NaCl. The temperature of 50% dissociation of bound oligodeoxynucleotide after being washed for 30 min was measured with a dot-blot assay. These results, together with the thermodynamic results, indicate that the substitution of methylphosphonate linkages at high salt only affects the reverse rate constant.     

Antisense inhibition of RNase P: mechanistic aspects and application to live bacteria

We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were approximately 10(5) m(-1) s(-1) with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growth-inhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg2+ ions.     

Methylphosphonate LNA: a locked nucleic acid with a methylphosphonate linkage

Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.     

4-(2-aminooxyethoxy)-2-(ethylureido)quinoline-oligonucleotide conjugates: synthesis,binding interactions,and derivatization with peptides

Oligo-2'-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2'-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 degrees C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2'-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ-oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ-oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.     

Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells

RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.     

Phosphoramidate analogues of DNA: synthesis and thermal stability of heteroduplexes

The deoxyoligonucleotide 5' AATCGGGCATGGATT (15-mer) was synthesized containing 12 phosphoramidate linkages derived from 2 primary and 2 secondary amines. The oligonucleotides were purified by reverse phase HPLC and characterized by PAGE. The thermal stability of the duplexes derived from these compounds, when hybridized to the complementary diester linked oligomer, were determined and compared to the diester and methanephosphonate oligomer. The results indicated that all analogue oligomers form less stable duplexes then the diester oligomer. A large difference was observed between primary and secondary amine derived phosphoramidates.     

Cleavage of RNA in hybrid duplexes by E. coli ribonuclease H. II. Substrate properties of nucleotides containing non-nucleotide linkers]

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for the E. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.     

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Cleavage of RNA in hybrid duplexes by theE. coli ribonuclease H: II. Substrate properties of oligonucleotides containing nonnucleotide linkers

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for theE. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2–1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3′-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3′-3′-phosphodiester bond at the 3′-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity. For the previous report, see [1].     

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.     

NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine.

The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the T m of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 degrees C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment.     

Oligonucleotide N3'-->P5' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3'-->P5' phosphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with complementary native phosphodiester DNA and exceptionally stable duplexes with RNA strands. Increases in duplex melting temperature, deltaTm, relatively to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modified nucleoside. Moreover, the phosphoramidate compounds form extremely stable triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of these triplexes usually exceed that of the isosequential phosphodiester counterparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nucleases both in vitro and in vivo. Oligonucleotide phosphoramidates apparently are cell permeable, and they have a good bioavailability and biodistribution, while being non-toxic in mice at therapeutically relevant doses. Duplexes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these compounds exerted high sequence-specific antisense activity in various cell lines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biological activity of these compounds relative to RNase H inducing oligonucleotides. Experimental results also indicate that oligonucleotide phosphoramidates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidate double stranded complexes can structurally mimic native RNA complexes, which could be efficiently and specifically recognized by the RNA binding proteins, such as HIV-1 Rev and Tat.