Trichloroacetaldehyde modified oligonucleotides

Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.     

Oligonucleotide charge reversal: 2'-O-lysylaminohexyl modified oligonucleotides

A novel cationic building nucleoside building block designed for antisense and siRNA oligonucleotides is presented. Protected L-lysine was coupled to 2'-O-aminohexyluridine and the resulting nucleoside was phosphitylated for automated oligonucleotide synthesis. An increasing number of these 2'-O-lysylaminohexyl nucleosides lowered the melting temperature of desoxy-thymidine homododecamers, but the decrease was lower than that for DNA/RNA hybrids. Incubation with an exonuclease showed the exceptionally high resistance against enzymatic degradation. CD spectrometry revealed a gradual transition towards an A-type oligonucleotide structure. Based on these data, the cationic building block is particularly suited for gapmer antisense as well as siRNA oligonucleotides.     

Novel method for the synthesis of 2' -phosphorylated oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2'-O-methylribonucleotides) that contain a 2'-phosphorylated ribonucleoside residue, and optimized it to avoid 2' -3' -isomerization and chain cleavage. Structures of the 2' -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2'-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.     

Targeted gene knockout by 2'-O-aminoethyl modified triplex forming oligonucleotides

Triplex forming oligonucleotides (TFOs) are of interest because of their potential for facile gene targeting. However, the failure of TFOs to bind target sequences at physiological pH and Mg(2+) concentration has limited their biological applications. Recently, pyrimidine TFOs with 2'-O-aminoethyl (AE) substitutions were shown to have enhanced kinetics and stability of triplex formation (Cuenoud, B., Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int. Ed. 37, 1288--1291). We have prepared psoralen-linked TFOs with varying amounts of the AE-modified residues, and have characterized them in biochemical assays in vitro, and in stability and HPRT gene knockout assays in vivo. The AE TFOs showed higher affinity for the target in vitro than a TFO with uniform 2'-OMe substitution, with relatively little loss of affinity when the assay was performed in reduced Mg(2+). Once formed they were also more stable in "physiological" buffer, with the greatest affinity and stability displayed by the TFO with all but one residue in the AE format. However, TFOs with lesser amounts of the AE modification formed the most stable triplexes in vivo, and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.     

Triplex-forming ability of modified oligonucleotides

We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA are particularly stable.     

Synthesis of amide-linked [(3')CH2CO-NH(5')] nucleoside analogues of small oligonucleotides

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3'-(carboxymethyl)-3'-deoxy- and 5'-amino-5'-deoxynucleoside derivatives provides amide dimers. Activated [3'-(carboxymethyl)-3'-deoxy] units with a 5'-azido-5'-deoxy function provide "masked" 5'-amino-5'-deoxy residues for chain extension, and a 5'-O-DMT-protected unit provides the 5'-terminus for attachment to a phosphodiester linkage.     

2'-bis-pyrene modified oligonucleotides: sensitive fluorescent probes of nucleic acids structure

A new type of fluorescent nucleic acid probes, 2-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2'-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2'-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.     

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2′-fluoro, 2′-O-propyl, 2′-O-methoxyethyl and 2′-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA–DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3°C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA–RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0°C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.     

Effect of nucleotide substitution on the peptidyltransferase activity of 2'(3')-O-(aminoacyl) oligonucleotides

Seven 2'(3')-O-(aminoacyl) trinucleotides with structures derived from the 3'-terminal C-C-A sequence of aa-tRNA via nucleotide substitutions were investigated as acceptor substrates in the peptidyltransferase reaction and as inhibitors of substrate binding to the peptidyltransferase A site. It was found that all tested compounds were active in both systems, although substitution in the first and second nucleotide position results in some decrease of acceptor activity. Remarkably, replacement of natural cytidylic acid residues in C-C-A-Phe with guanylic acid moieties resulted only in a small decrease of acceptor or binding activity. The results indicate that the acceptor sequence of aa-tRNA is not probably engaged in base pairing with a sequence of 23S RNA during its interaction with the peptidyltransferase A site.     

Enzymatic incorporation into oligonucleotides of modified nucleosides

Behaviour of modified nucleosides, tRNA components, and their analogues has been studied in the internucleotide bond formation catalysed by ribonucleases of various substrate specificity, polynucleotide phosphorylases, and T4 RNA ligase and the results are summarised in this paper. Pseudouridine, dihydrouridine, ribothymidine, 5-methylcytidine, inosine, and 6-methyladenosine can participate in the reaction of internucleotide bond formation the presence of most ribonucleases used, viz. Pb2, Pcl2, Pb1, Pch1, C2, T1, pancreatic RNase. 3-Methylcytidine and 4-acetylcytidine form internucleotide bond (as phosphate acceptors) usually by means of guanyl-specific ribonucleases, whereas 1-methylandenosine is incorporated with ribonuclease Pel2. 7-Methylguanosine and 1-methylguynosine 2',3'-cyclophosphates can be used as phosphate donors in the presence of ribonuclease Pb2; in the similar enzymatic reaction 6-isopentenyladenosine is an uneffective acceptor.     

Synthesis and properties of modified oligonucleotides.

Phosphorothioate oligonucleotide analogs conjugated to cholesteryl by a neutral, 6 atom linker are more effective inhibitors of HIV-1 in cell culture than the corresponding analogs conjugated via a phosphorothioate group. The antiviral activity correlates with the hydrophobic character of the oligonucleotide. Some new synthetic methodology is also discussed.     

Requirements for polyadenylation at the 3' end of LINE-1 elements

LINE-1 (L1) is the only active, autonomous, non-LTR, human retroelement. There are about 5 × 10⁵ L1 copies in the human genome, the majority of which are truncated at their 5′ ends. Both truncated and full-length L1 insertions contain a polyadenylation (polyA) signal at their 3′ ends. A typical polyA site consists of the three main cis-acting elements: a conserved hexamer, cleavage site, and a GU-rich downstream region. A newly inserted L1 copy contains the conserved AATAAA hexamer at the end of its sequence. However, the GU-rich downstream region has to be provided by the neighboring genomic sequences and therefore it would vary for every L1 copy. Using northern blot analysis of transiently transfected L1 expression vectors we demonstrate that L1 element contain sequence that allow efficient polyadenylation at the L1 3′ end upon retrotransposition into a new genomic location independent of the base composition downstream of the insertion site. The strategy of polyadenylation at the 3′ end of L1 parallels the approach the element employs at its 5′UTR by having an unusual internal polymerase II promoter, making new insertions less dependent on the properties of the flanking sequences at the new locus.     

Tuning of conformational preorganization in model 2',5'- and 3',5'-linked oligonucleotides by 3'- and 2'-O-methoxyethyl modification

Conformational properties of trimeric and tetrameric 2′,5′-linked oligonucleotides, 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2), and their 3′,5′-linked analogs, 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4), were examined with the use of heteronuclear NMR spectroscopy. The temperature-dependent ³J_HH, ³J_HP and ³J_CP coupling constants, acquired in the range of 273–343 K, gave insight into the conformation of sugar rings in terms of a two-state North ↔ South (N ↔ S) pseudorotational equilibrium and into the conformation of the sugar–phosphate backbone in the model antisense oligonucleotides 1–4. 2′,5′-linked oligomers 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) show preference for N-type conformers and indication of A-type conformational features, which is prerequisite for antisense hybridization. The drive of N ↔ S equilibrium in 1–4 has been rationalized with the competing gauche effects of 2′/3′-phosphodiester and 3′/2′-MOE groups, anomeric and steric effects. Furthermore, the pairwise comparisons of 3′-MOE with 3′-OH and 3′-deoxy 2′,5′-linked adenine trimers emphasized the fine tuning of N ↔ S equilibrium in 3′-MOE-A₃^2′,5′ (1) and 3′-MOE-A₄^2′,5′ (2) by the steric effects of 3′-MOE group and the possibility of water-mediated H-bonds with vicinal phosphodiester functionality. In full correspondence, the drive of N ↔ S equilibrium towards N by 2′-MOE in 3′,5′-linked analogs 2′-MOE-A₃^3′,5′ (3) and 2′-MOE-A₄^3′,5′ (4) is weaker in comparison with 3′-OH group in the corresponding ribo analogs. β^t, γ⁺ and ε^– rotamers are preferred in both 2′,5′- and in 3′,5′-linked oligonucleotides 1–4.     

Chemically modified oligonucleotides with efficient RNase H response

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.