ANTISENSE PROPERTIES OF 2′-O-DIMETHYLAMINOOXYETHYL (2′-O-DMAOE) OLIGONUCLEOTIDES

Antisense oligonucleotides with 2′-O-{2-[N,N-dimethyl)aminooxy]ethyl} or (2′-O-DMAOE) modification were synthesized and evaluated for nuclease resistance and pharmacology both in vitro and in vivo. This modification exhibits very high nuclease resistance and efficacy in various biological (ICAM-1, C-raf and PKC-α) targets.     

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.     

2′-DMAOE RNA: Emerging Oligonucleotides with Promising Antisense Properties

Abstract

Oligonucleotides with modifications at the carbohydrate 2′-position offer potential second-generation drug candidates¹. ISIS 13312, a chimeric compound targeting CMV retinitis, has 2′-O-methoxyethyl² (2′-MOE) modifications at the ends to offer enhanced binding affinity and nuclease resistance is an example of this trend. 2′-MOE modification offers high binding affinity and nuclease resistance presumably due to conformational constraints placed on the linkage by the oxygen-oxygen gauche effect³. On the other hand, 2′-O-aminopropyl modification (2′-AP) exhibits the highest nuclease resistance⁴, due to the presence of a cationic charge at the physiological pH. However, it lacks the binding affinity advantage of MOE due to the lack of oxygen-oxygen gauche effect. To optimize the antisense properties of both 2′-MOE and 2′-AP modifications, we have designed and constructed 2′-O-(aminooxyethyl) modification (2′-AOE)⁵ and 2′-O-(dimethylaminooxy ethyl) modification (2′-DMAOE) and synthesized oligomers having these modifications. 2′-DMAOE oligomers demonstrate higher binding affinity and nuclease resistance than 2′-MOE oligomers and stand out as promising candidates for future antisense oligonucleotide drug development.     

Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA.

2'-O-(2-Methoxyethyl)-RNA (MOE-RNA) is a nucleic acid analog with promising features for antisense applications. Compared with phosphorothioate DNA (PS-DNA), the MOE modification offers improved nuclease resistance, enhanced RNA affinity, improved cellular uptake and intestinal absorption, reduced toxicity and immune stimulation. The crystal structure of a fully modified MOE-RNA dodecamer duplex (CGCGAAUUCGCG) was determined at 1.7 A resolution. In the majority of the MOE substituents, the torsion angle around the ethylene alkyl chain assumes a gauche conformation. The conformational preorganization of the MOE groups is consistent with the improved RNA affinity and the extensive hydration of the substituents could play a role in the improved cellular uptake of MOE-RNA. A specific hydration pattern that bridges substituent and phosphate oxygen atoms in the minor groove of MOE-RNA may explain its high nuclease resistance.     

Intracellular stability of 2'-OMe-4'-thioribonucleoside modified siRNA leads to long-term RNAi effect

Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2'-OMe-4'-thioRNA, a hybrid chemical modification based on 2'-OMeRNA and 4'-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2'-OMe-4'-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT-PCR revealed that the intracellular stability of the siRNA modified with 2'-OMe-4'-thioribonucleosides contributed significantly to the duration of its RNAi activity.     

Inhibition of influenza virus RNA (PB2 mRNA) expression by a modified DNA enzyme.

DNA enzymes are RNA-cleaving single stranded DNA molecules. The structure and the catalytic domain of a DNA enzyme were determined by Santro et al. in 1997. In this study, we have designed several types of DNA enzymes (PB2Dz) targeted to the PB2 mRNA translation initiation region of influenza A virus, and examined their cleavage kinetics, nuclease resistance, and a luciferase gene reporter assay. Using a synthetic substrate, these DNA enzymes were shown to have cleavage activity that is dependent on the length of the substrate recognition domain. To confer serum nuclease resistance to the DNA enzymes, we designed a new type of DNA enzyme that has the N3'-P5' phosphoramidate modification (PB2Dz-N) at each terminal. We examined the activity of this DNA enzyme in vivo. The DNA enzymes used in this study inhibited the expression of the PB2-luciferase gene in COS cells. These results suggest that DNA enzymes are potentially useful as gene inactivating agents of influenza A virus.     

Synthesis and properties of novel 2'-O-alkoxymethyl-modified nucleic acids

Novel 2'-O-modified oligoribonucleotides with alkoxymethyl skeletons were synthesized, and their ability to hybridize complementary nucleic acids and their nuclease resistance were analyzed. The hybridization ability was improved by introducing electron-withdrawing groups and the increases in melting temperature (T(m) value) was particularly high for chlorine-substituted compounds. Nuclease resistance of these 2'-O-alkoxymethylated oligomers was lower than expected, but cyano substitution resulted in a higher nuclease resistance than 2'-O-methylation.     

寡聚脱氧核苷酸的结构与抗降解特性的研究

合成了４段具有不同高级结构或不同修饰的寡聚脱氧核苷酸,检查它们在２０％血清中的稳定性．发现：（１）寡核苷酸主要被血清中的３′外切核酸酶降解,未经修饰的线性寡核苷酸降解严重;（２）末端部分硫代修饰的寡核苷酸稳定性明显提高;（３）自身互补形成的配对结构可有效保护３′末端．具有４个以上（含４个）ＧＣ对的３′端发夹结构寡核苷酸,其抗核酸酶的能力几乎与硫代修饰的寡核苷酸相当．     

Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications

The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.     

G-quadruplex induced stabilization by 2'-deoxy-2'-fluoro-D-arabinonucleic acids (2'F-ANA)

The impact of 2'-deoxy-2'-fluoroarabinonucleotide residues (2'F-araN) on different G-quadruplexes derived from a thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), an anti-HIV phosphorothioate aptamer PS-d(T2G4T2) and a DNA telomeric sequence d(G4T4G4) via UV thermal melting (T(m)) and circular dichroism (CD) experiments has been investigated. Generally, replacement of deoxyguanosines that adopt the anti conformation (anti-guanines) with 2'F-araG can stabilize G-quartets and maintain the quadruplex conformation, while replacement of syn-guanines with 2'F-araG is not favored and results in a dramatic switch to an alternative quadruplex conformation. It was found that incorporation of 2'F-araG or T residues into a thrombin-binding DNA G-quadruplex stabilizes the complex (DeltaT(m) up to approximately +3 degrees C/2'F-araN modification); 2'F-araN units also increased the half-life in 10% fetal bovine serum (FBS) up to 48-fold. Two modified thrombin-binding aptamers (PG13 and PG14) show an approximately 4-fold increase in binding affinity to thrombin, as assessed via a nitrocellulose filter binding assay, both with increased thermal stability (approximately 1 degrees C/2'F-ANA modification increase in T(m)) and nuclease resistance (4-7-fold) as well. Therefore, the 2'-deoxy-2'-fluoro-d-arabinonucleic acid (2'F-ANA) modification is well suited to tune (and improve) the physicochemical and biological properties of naturally occurring DNA G-quartets.     

Biological availability and nuclease resistance extend the in vitro activity of a phosphorothioate-3'hydroxypropylamine oligonucleotide.

Augmented biological activity in vitro has been demonstrated in oligonucleotides (oligos) modified to provide nuclease resistance, to enhance cellular uptake or to increase target affinity. How chemical modification affects the duration of effect of an oligo with potent activity has not been investigated directly. We postulated that modification with internucleotide phosphorothioates and 3' alkylamine provided additional nuclease protection which could significantly extend the biological activity of a 26 mer, (T2). We showed this analog, sT2a, could maximally inhibit interferon gamma-induced HLA-DR mRNA synthesis and surface expression in both HeLa and retinal pigmented epithelial cells and could continue to be effective, in the absence of oligo, 15 days following initial oligo treatment; an effect not observed with its 3'amine counterpart, T2a. In vitro stability studies confirmed that sT2a conferred the greatest stability to nucleases and that cellular accumulation of 32P-sT2a in both cell types was also greater than other T2 oligos. Using confocal microscopy, we revealed that the intracellular distribution of sT2a favored greater nuclear accumulation and release of oligo from cytoplasmic vesicles; a pattern not observed with T2a. These results suggest that phosphorothioate-3'amine modification could increase the duration of effect of T2 oligo by altering nuclease resistance as well as intracellular accumulation and distribution; factors known to affect biological availability.     

2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles

Oligonucleotides modified with 2 ',4 '-BNA(NC) (N-H)/(N-Me) monomers exhibited excellent hybridizing and nuclease resistance properties. Duplex and triplex thermal stabilities were greatly enhanced by incorporating 2',4'-BNA(NC) (N-H) and (N-Me) monomers and nuclease resistance was tremendously higher than that of natural oligonucleotide.     

Preferential Resistance of Phosphodiester Bonds between Deoxycytidine and 5'-Adjacent Bases to Chlamydomonas Nuclease C

Chlamydomonas Ca²⁺-dependent nuclease (nuclease C) has beenshown to be polymorphic (Ogawa and Kuroiwa 1985a). Aged preparationsof crude extract obtained in the absence of protease inhibitorcontained little nuclease C1&2. Instead, most of the activitiesappeared in association with smaller molecules similar in sizeto nuclease C3 and C4 that are members of nuclease C isozymes,suggesting that the polymorphism may be caused by the actionof an endogenous protease. Both the original form and the active fragments showed base-specificendo-exonucleolytic activity, and liberated an extremely lowlevel of 3'-dCMP as compared with the other three 3'-dNMPs.We compared the extent of hydrolysis of phosphodiester bondsbetween two bases of all possible combinations. The result showedpreferential resistance of phosphodiester bonds between dC and5' -adjacent bases to nuclease C. This explains the paucityof 3'-dCMP in the digests of DNA. The base-specific action ofnuclease C1&2 was preserved during the modification by endogenousprotease. (Received September 6, 1986; Accepted December 23, 1986)     

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.     

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.     

2'-O-methyl-modified anti-MDR1 fork-siRNA duplexes exhibiting high nuclease resistance and prolonged silencing activity

The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.     

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4′-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4′-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s²U) and 5-methyl-2-thiouridine (m⁵s²U) are natural nucleotide analogs. s²U in tRNA confers greater specificity of codon–anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2′-deoxyribose sugars for a C3′-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2′-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2′-O-[2-(methoxy)ethyl]-2-thiothymidine (m⁵s²Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m⁵s²Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m⁵Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter ‘opening’, an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m⁵s²Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2′-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.     

2'-carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation

The 2'-position of the carbohydrate moiety has proven to be a fertile position for oligonucleotide modifications for antisense technology. The 2'-modifications exhibit high binding affinity to target RNA, enhanced chemical stability and nuclease resistance and increased lipophilicity. All high binding affinity 2'-modifications have C3'-endo sugar pucker. In addition to gauche effects, charge effects are also important in determining the level of their nuclease resistance. Pharmacokinetic properties of oligonucleotides are altered by 2'-conjugates. For certain modifications (e.g., 2'-F), the configuration at the 2'-position, arabino vs. ribo, determines their ability to activate the enzyme RNase H.     

Effects of vinylphosphonate internucleotide linkages on the cleavage specificity of exonuclease III and on the activity of DNA polymerase I

We have previously reported the synthesis of vinylphosphonate-linked thymidine dimers and their incorporation into synthetic oligonucleotides to create vinylphosphonate internucleotide linkages in the DNA. Such linkages have a profound effect on DNA backbone rotational flexibility, and we have shown that the PcrA helicase, which requires such flexibility, is inhibited when it encounters these linkages on the translocating strand. In this study, we have investigated the effects of these linkages on the dsDNA specific exonuclease III and on the ssDNA specific mung bean nuclease to establish whether our modification confers resistance to nucleases making it suitable for antisense therapy applications. We also investigated the effect on DNA polymerase I to establish whether we could in the future use this enzyme to incorporate these linkages in the DNA. Our results show that a single modification does not affect the activity of DNA polymerase I, but four vinylphosphonate linkages in tandem inhibit its activity. Furthermore, such linkages do not confer significant nuclease resistance to either exonuclease III or mung bean nuclease, but unexpectedly, they alter the cleavage specificity of exonuclease III.