Specific Base Pairs Research Articles

Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements.

Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposition is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic distribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydrogen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements.

Roles of stiffness and excluded volume in DNA denaturation.

The nature and the universal properties of DNA thermal denaturation are investigated by Monte Carlo simulations. For suitable lattice models we determine the exponent c describing the decay of the probability distribution of denaturated loops of length l, P approximately l(-c). If excluded volume effects are fully taken into account, c = 2.10(4) is consistent with a first order transition. The stiffness of the double stranded chain has the effect of sharpening the transition, if it is continuous, but not of changing its order and the value of the exponent c, which is also robust with respect to inclusion of specific base-pair sequence heterogeneities.

Labeling fluorescence in situ hybridization probes for RNA targets.

Ribonucleic acids (RNA) can be detected in cellular specimens by fluorescence in situ hybridization (FISH) using labeled RNA, deoxyribonucleic acid (DNA), or nucleic acids analogs. The RNA targets are often messenger RNA (mRNA) molecules that have been transcribed from the genomic DNA. Specific mRNA sequences can be selectively identified out of the many co-expressed mRNA molecules owing to the high specificity of nucleic acid base pairing. The analysis of selected mRNA sequences is an important measure of many cellular properties, including, for example, the identification of specific cell types, the identification of different stages of cell differentiation, and measuring abnormal mRNA expression, as often accompanies cancer development. RNA probes can be directly conjugated with fluorescent labels, or conjugated with labels that specifically associate with fluorescent compounds. Once hybridized, the presence and relative abundance of an RNA target can be qualitatively assessed visually based on the fluorescence intensity, or in a more quantitative fashion, by digital imaging and image analysis.

Analysis of promoter methylation and its role in silencing metallothionein I gene expression in tumor cells

Publisher Summary A method exists to identify the methylated cytosines at specific sites within a gene. Bisulfite genomic sequencing is an innovative technique that can be used effectively to determine the methylation status of specific CpG base pairs in a specific gene. Briefly, the procedure consists of denaturation, sulfonation, desulfonation, and polymerase chain reaction (PCR) amplification followed by restriction enzyme digestion and sequencing. Treatment of genomic DNA with bisulfite reagent under denaturing conditions converts cytosines in the DNA to uracils, amplified as thymine during subsequent PCR. Therefore, the few remaining cytosines in the sequencing gel are due to either 5-methylcytosine in the chromosomal DNA or incomplete bisulfite conversion, a common problem in the reaction. To overcome the artifact the original protocol has been modified. Denaturing DNA at 95° for 2 min after 30 min of desulfonation during this procedure facilitates completion of the reaction. The amplified DNA is digested with several restriction enzymes before sequencing to check complete bisulfite conversion.

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.

Using base-specific Salmonella tester strains to characterize the types of mutation induced by benzidine and benzidine congeners after reductive metabolism

Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3′-dichlorobenzidine HCl (DCBz), 3,3′-dimethylbenzidine (DMBz), 3,3′-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present. Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations. Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested. Using the base-specific tester strains of S. typhimurium (TA7001–TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism. Bz was weakly mutagenic in TA7005 (0.04 revertants/μg). ABP was mutagenic in TA7002 (1.4 revertants/μg), TA7004 (0.6 revertants/μg), TA7005 (2.98 revertants/μg) and TA7006 (0.4 revertants/μg). DCBz was weakly mutagenic in TA7004 (0.01 revertants/μg). It was concluded that benzidine induced some CG->AT transversions in addition to frameshift mutations. ABP induced TA->AT, CG->AT, and CG->GC transversions as well as GC->AT transitions. DCBz induced only GC->AT transitions. Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

Aromatic extended bisamidines: synthesis, inhibition of topoisomerases, and anticancer cytotoxicity in vitro.

A series of four aromatic extended bisamidines (12-15) differing in the nature of their terminal basic side chains were synthesized and evaluated for cytotoxic activity in MCF-7 cultured breast cancer cells. The concentrations of 12, 13, 14, and 15 needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) were found to be 63 microM, 85 microM, 77 microM, and 97 microM, respectively. To test whether cytotoxic properties were related to DNA-binding and topoisomerase action, the bisamidines 12-15 were evaluated in a cell-free system. Data from the ethidium displacement assay showed that bisamidines 12-15 have significant affinity for DNA and show moderate specificity for AT base pairs. In the topoisomerase II assay, the relaxation of DNA was inhibited with all four drugs and the extent of inhibition was directly proportional to the drug concentration. This suggests that DNA binding may be implicated in the cytotoxicity of these bisamidines, possibly by inhibiting interactions between topoisomerase II and their DNA targets.

Aromatic Extended Bisamidines: Synthesis, Inhibition of Topoisomerases, and Anticancer Cytotoxicity in Vitro

A series of four aromatic extended bisamidines (12-15) differing in the nature of their terminal basic side chains were synthesized and evaluated for cytotoxic activity in MCF-7 cultured breast cancer cells. The concentrations of 12, 13, 14, and 15 needed to inhibit [3H]thymidine incorporation into DNA by 50% (IC50) were found to be 63 microM, 85 microM, 77 microM, and 97 microM, respectively. To test whether cytotoxic properties were related to DNA-binding and topoisomerase action, the bisamidines 12-15 were evaluated in a cell-free system. Data from the ethidium displacement assay showed that bisamidines 12-15 have significant affinity for DNA and show moderate specificity for AT base pairs. In the topoisomerase II assay, the relaxation of DNA was inhibited with all four drugs and the extent of inhibition was directly proportional to the drug concentration. This suggests that DNA binding may be implicated in the cytotoxicity of these bisamidines, possibly by inhibiting interactions between topoisomerase II and their DNA targets.

A Novel Brain-specific Box C/D Small Nucleolar RNA Processed from Tandemly Repeated Introns of a Noncoding RNA Gene in Rats

Antisense box C/D small nucleolar RNAs (snoRNAs) guide the 2'-O-ribose methylations of eukaryotic rRNAs and small nuclear RNAs (snRNAs) through formation of a specific base pairing at each RNA methylation site. By analysis of a box C/D snoRNA cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to rRNA or an snRNA and exhibits a brain-specific expression pattern. It is uniformly expressed in all major areas of adult rat brain (except for choroid plexus) and throughout rat brain ontogeny but exclusively detected in neurons in which it exhibits a nucleolar localization. In vertebrates, known methylation guide snoRNAs are intron-encoded and processed from transcripts of housekeeping genes. In contrast, RBII-36 snoRNA is intron-encoded in a gene preferentially expressed in the rat central nervous system and not in proliferating cells. Remarkably, this host gene, which encodes a previously reported noncoding RNA, Bsr, spans tandemly repeated 0.9-kilobase units including the snoRNA-containing intron. The novel brain-specific snoRNA appears to result not only from processing of the debranched lariat but also from endonucleolytic cleavages of unspliced Bsr RNA (i.e. an alternative splicing-independent pathway unreported so far for mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA were exclusively detected in the Rattus genus of rodents, suggesting a very recent origin of this brain-specific snoRNA.

Mutations at Position −10 in the λ PR Promoter Primarily Affect Conversion of the Initial Closed Complex (RPc) to a Stable, Closed Intermediate (RPi)

The effects of mutations of --10 T:A to A:T, C:G, or G:C in the lambda P(R) promoter on formation of transcriptionally competent open complexes were studied by DNAse I footprinting, KMnO(4)-sensitivity, and abortive initiation kinetic analysis. The mutations --10A (T:A --> A:T) and --10C significantly reduce k(f), the composite rate constant for conversion of closed complexes (RP(c)) to open complexes (RP(o)) but do not affect K(B), the equilibrium constant for formation of closed complexes. Unlike the other mutants or wild-type P(R), the mutation with the largest effect on open complex formation, --10G (T:A --> G:C), substantially decreases the occupancy of the promoter. When reduced occupancy is taken into account, the calculated effect of the mutation on k(f) is a 20-fold reduction. Analysis of open complex formation by a three-step pathway that includes an additional intermediate, RP(i), indicates that the primary effect of all three mutations is a reduction in the rate of isomerization of RP(c) to RP(i), which precedes DNA strand separation. Thus, RNA polymerase holoenzyme must recognize specific base pairs in the --10 region of P(R) while the DNA is still double-stranded. Comparison of the observed level of stable complexes (RP(i) plus RP(o)) with the level of productive complexes (RP(o)) indicates that the --10G mutation may also affect the equilibrium between RP(i) and RP(o) at 37 degrees. Open complexes formed at the three mutant promoters are approximately 3-5 times less stable at 37 degrees than those formed at wild-type P(R).

DNA-binding activity and cytotoxicity of the extended diphenylfuran bisamidines in breast cancer MCF-7 cells.

The DNA binding properties of three novel extended diphenylfuran bisamidines (1-3) possessing different dicationic terminal side chains were studied. The ultrafiltration assay showed that bisamidines 1-3 have significant affinity for DNA. The DNA-binding data for bisamidines 1-3 using homopolymers poly(dA-dT)- poly(dA-dT) and poly(dG-dC)- poly(dG-dC), indicated that these compounds show moderate specificity for AT base pairs. We studied the cytotoxicity effects of bisamidines 1-3, Hoechst 33258 and DAPI (4',6-diamidino-2-phenylindole) in cultured breast cancer MCF-7 cells. The bisamidines 1-3 showed comparable antitumour activity to Hoechst 33258, but were substantially more cytotoxic compared to DAPI. These data show that in broad terms the cytotoxic potency of bisamidines 1-3 in cultured breast cancer MCF-7 cells decreases with the size of the alkyl group substituent (cyclopropyl>isopropyl>cyclopentyl), in accord with their increases in DNA affinity, as shown by the binding constant values.

In vitro selection of a novel nuclease-resistant RNA phosphodiesterase

Background: Ribonucleotide-based enzymes (ribozymes) that cleave pathological RNAs are being developed as therapeutic agents. Chemical modification of the hammerhead ribozyme has produced nuclease-resistant catalysts that cleave targeted mRNAs in cell culture and exhibit antitumor activity in animals. Unfortunately, stabilizing modifications usually reduce the catalytic rate in vitro. An alternative to rationally designed chemical modifications of existing ribozymes is to identify novel motifs through in vitro selection of nuclease-stable sequence space. This approach is desirable because the catalysts can be optimized to function under simulated physiological conditions. Results: Utilizing in vitro selection, we have identified a nuclease-stable phosphodiesterase that demonstrated optimal activity at simulated physiological conditions. The initial library of 10 14 unique molecules contained 40 randomized nucleotides with all pyrimidines in a nuclease-stabilized 2′-deoxy-2′-amino format. The selection required trans-cleaving activity and base-pairing specificity towards a resin-bound RNA substrate. Initial selective pressure was permissive, with a 30 min reaction time and 25 mM Mg 2+. Stringency of selection pressure was gradually increased until final conditions of 1 mM Mg 2+ and less than 1 min reaction times were achieved. The resulting 61-mer catalyst required the 2′-amino substitutions at selected pyrimidine positions and was stable in human serum (half-life of 16 h). Conclusions: We demonstrated that it is possible to identify completely novel, nuclease-resistant ribozymes capable of trans-cleaving target RNAs at physiologically relevant Mg 2+ concentrations. The new ribozyme motif has minimal substrate requirements, allowing for a wide range of potential RNA targets.

Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents

There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites, in addition to sites characteristic of untargeted mustards.

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Effects of rho-independent terminators on the expression of the prokaryoticβ-glucuronidase gene in tobacco

The threonine (thr) attenuator with a dyad symmetrical structure is from the regulatory region of the thr operon of Escherichia coli, and encodes RNA with a stem-and-loop structure followed by a stretch of uridine residues. The thr attenuator and its variants were subcloned into the region between the 35S promoter and β-glucuronidase (GUS) coding region, and the transient expression of GUS gene in tobacco protoplast was treated as a reporter for gene regulation in plants. Results from the 14 variants in the stem region of the thr attenuator indicated that both base pairing and sequence specificity in the G+C-rich region of the stem were important for the GUS expression, but 1 base mismatch in the A+U-rich region of stem did not affect the GUS expression in plants. Seven variants with nested deletion in the stretch of uridine residues were also analyzed, and the results suggested that the variants with the shorter uridine stretch produced more GUS protein than those with the longer stretch. Transgenic tobacco plants with the thr attenuator and its variants located between the 35S promoter and GUS coding region were also generated, and their steady state RNAs were hybridized with 2 radioactive antisense RNA probes which bound 5′ and 3′ of the thr hairpin, respectively. After the digestion of S1 nuclease, the amount of the nuclease-resistant transcript from the protection of the 5′ antisense RNA probe was much more than that from the protection of the 3′ probe in all tested variants. This result suggests that these dyad symmetries may affect transcription of plant RNA polymerase II.

RNA solvation: a molecular dynamics simulation perspective.

With the availability of accurate methods to treat the electrostatic long-range interactions, molecular dynamics simulations have resulted in refined dynamical models of the structure of the hydration shell around RNA motifs. The models reviewed here range from basic Watson-Crick to more specific noncanonical base pairs, from "simple" double helices to RNA molecules displaying more complex tertiary folds, and from DNA/RNA hybrid double helices to RNA hybrids formed with a chemically modified strand.

Adenine−Uridine Base Pairing at the Water−Solid-Interface

The formation of the base pair adenine-uracil at a water−solid interface, at an immobilized monolayer of adenine disulfide with adenine groups exposed to the very surface, respectively, is shown here. To overcome the steric hindrance of tightly packed adenine groups in a pure adenine thiolate monolayer on gold, the formation of self-assembled monolayers out of a binary mixture of the adenine disulfide and CH3- or OH-terminated thiols are investigated. Electro-chemical investigations, surface plasmon spectroscopy (PSP, plasmon surface polariton), multimode waveguide-PSP-coupling spectroscopy, contact angle measurements, and spontaneous desorption time-of-flight mass spectrometry were used to characterize the monolayers. The specific base pairing was investigated for a variety of monolayer compositions. A specific base pairing was successful for an optimized mixed adenine/OH-terminated thiol monolayer. Nevertheless unspecific binding is a problem.

DNA Binding Mode and Sequence Specificity of Piperazinylcarbonyloxyethyl Derivatives of Anthracene and Pyrene

Four novel piperazinylcarbonyloxyethyl derivatives of anthracene and pyrene have been prepared and investigated with respect to sequence specificity and synergism between hydrophobic and electrostatic effects upon binding to DNA. Linear and circular dichroism spectroscopy was used to assess the orientation of the aromatic chromophores relative to the nucleobases. Anthracene and pyrene derivatives 2a and 3 are both concluded to bind to homo-polynucleotide poly(dA-dT)(2) by intercalation of their aromatic moieties between base pairs, with a binding constant K-AT of 4 x 10(5) M-1 and 2 x 10(6) M-1, respectively. Significantly reduced affinities (K-GC = 3 x 10(4) M-1 and 10(5) M-1, respectively) are observed with poly(dG-dC)(2), due to less favorable interactions of the piperazinium tail in the minor groove. Base pair specificity is reflected in the binding thermodynamics, with the binding to AT being more enthalpically driven than the binding to GC. Phenyl substitution at the quaternary piperazinium site of the anthracene derivative 2b, does not affect the ratio K-AT/K-GC, but reduces the affinity for both AT End GC slightly. Moreover, the phenyl group in the 10-position of 4 prevents intercalation, and apparently, this compound binds externally to both AT and GC duplex polynucleotides. The results are discussed in terms of general features of the interactions of the intercalating and minor-groove binding molecular moieties, and their interplay with each other, with potentials for tuning specificity.

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron.

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.

Chiral and steric effects in the efficient binding of alpha-anomeric deoxyoligonucleoside N-alkylphosphoramidates to ssDNA and RNA.

We report hybridization properties of new phosphate-modified alpha-oligonucleoside analogs with non-ionic or cationic internucleotide linkages such as methoxy-ethylphosphoramidate (PNHME), phosphoromorpholi-date (PMOR) and dimethylaminopropylphosphor-amidate (PNHDMAP). First we evaluated the chirality effect of the phosphorus atom on the affinity of alpha- or beta-dodecanucleoside phosphodiesters containing one chirally enriched N -alkylphosphoramidate linkage located in the middle of the sequence d(TCTT-AA*CCCACA). As for P-substituted beta-oligonucleo-tides, a difference in binding behavior between the two diastereoisomers (difference in Delta T (m)) exists in the hybridization properties of alpha-analogs when DNA was the target but this effect was not detrimental to duplex stability. This effect was considerably reduced when RNA was the target. Secondly we studied the effect of steric hindrance around phosphorus on the affinity of fully modified beta- and alpha-oligonucleoside N -alkylphosphoramidates for their DNA and RNA targets. This effect was very weak with alpha-analogs whereas it was more pronounced with beta-oligos. PNHME-modified alpha-oligonucleosides formed more stable duplexes with DNA (Delta T (m)+9.6 degrees C) and RNA (Delta T (m)+1.4 degrees C) targets than the 'parent' phosphodiester. Finally, base pairing specificity of these alpha-oligonucleo-side N -alkylphosphoramidates for their targets was found to be as high as for natural oligonucleoside phosphodiesters.