Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides

The antisense approach is conceptually simple and elegant; to design an inhibitor of a specific mRNA, one needs only to know the sequence of the targeted mRNA and an appropriately modified complementary oligonucleotide. Of the many analogs of oligodeoxynucleotides explored as antisense agents, phosphorothioate analogs have been studied the most extensively. The use of phosphorothioate oligodeoxynucleotides as antisense agents in various studies have shown promising results. However, they have also indicated that quite often, biological effects observed could be solely or partly non-specific in nature. It is becoming clear that not all phosphorothioate oligodeoxynucleotides of varying length and base composition are the same, and important consideration should be given to maintain antisense mechanisms while identifying effective antisense oligonucleotides. In this review, I have summarized the progress made in my laboratory in understanding the specificity and mechanism of actions of phosphorothioate oligonucleotides and the rationale for designing second-generation mixed-backbone oligonucleotides.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

A new primer for sequencing DNA fragments cloned in M13mp vectors

Tridecadeoxyribonucleotide d(CCAGGGTTTTCCC) was prepared by solid phase crown-ether-catalyzed phosphotriester method and proposed a new sequencing primer. This primer expands capacities of the Sanger dideoxy-chain-terminating method to solve various sequencing problems as compared to well-known universal primers. Criteria of the choice of an oligonucleotide primer without computer analysis of nucleotide sequence are described.     

Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin

Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements. These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., and Gamper, H. B. (1996) Biochemistry 35, 11170-11176). Antisense ODNs with similar properties should be less encumbered by RNA secondary structure. Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs. Optimal binding was observed when the entire hairpin sequence was targeted. Translation of L. amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs. Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro. SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.     

Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages.

The replication of human immunodeficiency viruses (HIV) in human macrophages is influenced by genetic determinants which have been mapped predominantly to the viral envelope. However, in HIV-2, the vpr gene has also been suggested as an important modulator of viral expression in human macrophages. We synthesized five antisense phosphorothioate oligodeoxynucleotides complementary to the vpr mRNA of HIV-1Ba-L, a highly macrophage-tropic viral strain, and measured their effect on HIV-1Ba-L replication in primary human macrophages. All of the oligodeoxynucleotides displayed some level of non-sequence-specific inhibition of viral replication; however, only the antisense one had an additional effect on viral production in primary macrophages. Of the five antisense oligodeoxynucleotides tested, only one did not show any additional effect on viral production, whereas all the others inhibited viral replication to a similar degree (70 to 100%). Variation in the degree of inhibition was observed by using five different donors of human primary macrophages. The phosphorothioate oligonucleotides, targeted to the initiating methionine of the Vpr protein, had an inhibitory effect at both 20 and 10 microM only when the size was increased from 24 to 27 bases. Thus, HIV-1 replication in human macrophages is modulated by the expression of the vpr gene, and it is conceivable that vpr antisense oligodeoxynucleotides could be used in combination with antisense oligodeoxynucleotides against other HIV-1 regulatory genes to better control viral expression in human macrophages.     

A method of determining the primary structure of oligodeoxyribonucleotides

Sanger method was modified to fulfill the requirements of sequencing of oligodeoxyribonucleotides. E. coli DNA polymerase I Klenow fragment was used for all the reactions. The method consists of three steps made in succession in one tube: 1. Optional hydrolysis of a 5'-labeled oligodeoxyribonucleotide primer in order to get a set of primers of different lengths. 2. Elongation of the produced set of primers in the presence of a template, natural dNTPs and chain terminating dNTP analogs. 3. Hydrolysis of the products of the previous step in order to remove the unterminated molecules. Change of steps in achieved just by varying the reaction conditions without any product purification. The method in insensitive to the presence of admixture of oligonucleotides which is not complementary to the primer or to the template.     

Oligodeoxynucleotide analogs as informational drugs to regulate translation.

Of the chemically modified backbone analogs of oligodeoxynucleotides that have been developed for antisense applications, the phosphorothioate (PS) analog has perhaps the best properties. Nevertheless, it also has certain disadvantages, notably reduced hybridization and increased non-selective inhibition of translation, compared to the natural phosphodiester (PO) compounds. We have therefore synthesized and characterized a series of co-polymers, with the same antisense beta-globin sequence, but with different repeated sequences of PO and PS and tested them for their comparative properties. The results indicate that a PO-PS co-polymer is the best backbone modification for an antisense compound.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.     

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

Oligodeoxynucleotide analogs with 5'-linked anthraquinone

We report here the synthesis of novel 5'-linked oligodeoxynucleotides, both normal phosphodiester and phosphorothioate analogs, in which a covalently attached group at the 5'-terminus is an anthraquinone. These compounds represent a new class of antisense compounds in which the base sequence of the oligodeoxynucleotide serves to deliver a nuclease-resistant reactive drug-like molecule to a cellular target nucleic acid (mRNA or DNA).     

Chimeric RNA and 2'-O, 4'-C-ethylene-bridged nucleic acids have stronger activity than phosphorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA

Antisense phosphorothioate oligodeoxynucleotides against exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame. Because phosphorothioate oligonucleotides are associated with a variety of toxic nonantisense effects, several modifications of nucleic acid have been introduced to alleviate this toxicity. Recently, a 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA trade mark, Sankyo Lifetech Co., Ltd., Tokyo, Japan) was reported to have high affinity to complementary RNA strands and be resistant to nuclease digestion. Here, we examined the ability of this modified nucleic acid to induce exon skipping. Oligonucleotides having the same sequence as the phosphorothioate oligonucleotides but with some stretches of modified backbone (2'-O-methyl RNA with an ENA5-mer at the 5'-end and 3'-end) (RNA/ENA chimera) were transfected into myocytes, and the expressed dystrophin mRNA was analyzed. The RNA/ENA chimera induced exon 19 skipping in a dose-dependent and time-dependent manner. Remarkably, the exon 19-skipping activity of the RNA/ENA chimera was more than 40 times stronger than that of the corresponding conventional phosphorothioate oligodeoxynucleotide. This is the first report of such strong activity of an RNA/ENA chimera in the induction of exon skipping in the dystrophin gene. This new technology will allow the development of less toxic antisense drugs, making long-term therapy possible.     

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.     

Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro

We have investigated the mechanism of frameshift (deletion) mutagenesis induced by acetylaminofluorene- (AAF-) derived DNA adducts. dG-AAF-modified oligodeoxynucleotides, with different bases positioned 5' to the lesion, were annealed to (32)P-labeled 13-mer primers and then used in primer extension reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. When the dNMP positioned opposite dG-AAF could pair with its complementary base at the 5' flanking position, single-base deletions were produced at high frequency. Similarly, when the complementary base was two positions 5' to the dG-AAF, two-base deletions occurred. The relative frequency of base insertions opposite dG-AAF followed the order dCMP > dAMP > dGMP > dTMP; the frequency of dNTP insertion opposite the lesion paralleled the formation of frameshift deletions. When a template designed to induce three-base deletions was used for translesion synthesis catalyzed by the exo(-) Klenow fragment, the expected three-base deletion was formed. When dG-AAF-modified templates containing iterated bases 5' to the lesion were annealed to primers with the complementary dNMP positioned opposite the lesion, the dNMP inserted opposite the dG-AAF tended to pair with the complementary base 5' to the lesion, thereby forming shorter deletions. Taken together, these results support the molecular mechanism for frameshift deletion proposed earlier by Shibutani and Grollman in which direct base insertion precedes misalignment [(1993) J. Biol. Chem. 268, 11703].     

Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.