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PubMed2005

Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs.

RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.

Gene Silencing↗

PubMed1997

Crystal structures of A-DNA duplexes.

All crystal structures of A-DNA duplexes exhibit a typical crystal packing, with the termini of one molecule abutting the shallow grooves of symmetry related neighbors, while all other forms (B, Z, and RNA) tend to form infinitely stacked helices. The A-DNA arrangement leads to the formation of shallow groove base multiples that have implications for the structure of DNA in compacted states. The characteristic packing leaves big solvent channels, which can be sometimes occupied by B-DNA duplexes. Comparisons of the structures of the same oligomer crystallizing in two different space groups and of different sequences crystallizing in the same space group show that the lattice forces dominate the A-DNA conformation in the crystals, complicating the effort to elucidate the influence of the base sequence on the structures. Nevertheless, in both alternating and nonalternating fragments some sequence effects can still be uncovered. Furthermore, several studies have started to define the minimal sequence changes or chemical modifications that can interconvert the oligomers between different double-helical conformers (A-, B-, and Z-form). Overall, it is seen that the rigid nucleotide principle applies to the oligomeric fragments. Besides the structures of the naked DNAs, their interactions with water, polyamines, and metal ions have attracted considerable attention. There are conserved patterns in the hydration, involving both the grooves and the backbone, which are different from those of B-DNA or Z-DNA. Overall, A-DNA seems to be more economically hydrated than B-DNA, particularly around the sugar-phosphate backbone. Spermine was found to be able to bind exclusively to either of the grooves or to the phosphate groups of the backbone, or exhibit a mixed binding mode. The located metal cations prefer binding to guanine bases and phosphate groups. The only mispairs investigated in A-DNA are the wobble pairs, yielding structural insight into their effects on helix stabilities and hydration. G.T wobble pairs have been determined in various sequence contexts, where they differentially affect the conformations and stableness of the duplexes. The structure of a G.m5C base pair, which surprisingly also adopted the wobble conformation, suggests that a similar geometry may transiently exist for G.C pairs. These results from the crystalline state will be compared to the solution state and discussed in relation to their relevance in biology.

Base Sequence↗

PubMed1998

Triple helix-forming oligonucleotides which make imperfect Watson-Crick duplexes that compete with the creation of the triplex.

Because of the repetitive nature of the sequence, when titrating a G,A-rich, triple helix-forming oligonucleotide (TFO) with increasing concentrations of target duplex in order to obtain the dissociation constant of the complex, a duplex is sometimes first generated at intermediate concentrations of the target. This duplex is based on an imperfect Watson-Crick pairing of the TFO to the pyrimidine-rich strand of the target. An explanation is proposed for this duplex being obtained only in a certain domain of the titration range, before the triple helix becomes predominant.

Base Pairing↗

PubMed2002

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

Cross-Linking Reagents↗

PubMed1995

DNA duplexes containing methylated bases or non-nucleotide inserts in the recognition site are cleaved by restriction endonuclease R.EcoRII in presence of canonical substrate.

DNA duplexes containing the natural methylated bases N6-methyladenine (m6Ade), N4-methylcytosine (m4Cyt) or C5-methylcytosine (m5Cyt) in one strand of the recognition sequence are resistant to EcoRII restriction endonuclease (R.EcoRII). Hydrolysis of these modified duplexes was observed in the presence of the canonical substrate. Incorporation of m4Cyt or m5Cyt into both strands of the recognition sequence precludes such activation by a canonical substrate. R.EcoRII also fails to cleave substrate analogs in which one of the nucleosides in the recognition site is replaced by the 1,2-dideoxyribose (D) or by 1,3-propanediol (Prd) (modeling DNA with an abasic site). The hydrolysis of DNA duplexes with non-nucleotide inserts is also activated in the presence of canonical substrate. Thus, the two-substrate mechanism of EcoRII-DNA interaction allows hydrolysis of apurinic/apyrimidinic and hemimethylated DNA.

5-Methylcytosine↗

PubMed1999

Use of triplex-forming oligonucleotides and adenoviral constructs for studying the regulation of gene expression.

Short synthetic homopyrimidine- or homopurine-rich oligonucleotides can form sequence-specific triplexes with corresponding homopurine-homopyrimidine sites on duplex DNA and block transcription of a target gene in vitro. Such triplex-forming oligonucleotides (TFOs) can be rationally designed to target homopurine/homopyrimidine sequences that are often found in eukaryotic genes and thus used to modulate the expression of these genes. The antigene strategy using TFOs has been successfully applied to a number of genes in vitro. In this article we describe methods used in applying this antigene approach to the rat aquaporin 5 (rAQP5) gene. We specifically focus on the selection of TFOs based on the sequence of the target gene and on a novel method employing adenoviruses for delivery of TFOs to cells in vitro.

Adenoviridae↗

PubMed1980

DNA sequence of baboon highly repeated DNA: evidence for evolution by nonrandom unequal crossovers.

A highly repeated DNA was isolated from the West African baboon (Papio papio) as a 343-base-pair fragment after digestion of total baboon DNA with the restriction endonuclease BamHI. The DNA sequence of this fragment was obtained by chemical cleavage methods and is compared with the DNA sequence of related highly repeated primate DNAs from African green monkey (Cercopithecus aethiops) and man. The 343-base-pair baboon repeat consists of two related but nonidentical wings of 172 and 171 base pairs, respectively. The baboon 172-base-pair wing shares more homology with the African green monkey 172-base-pair repeat than with the baboon 171-base-pair wing. Comparison with the previously published monkey and human DNA sequences indicates that: (i) All the DNA sequences apparently arose from a common ancestral sequence. (ii) Evolution of the primate DNA sequences can be explained by a model involving unequal crossovers at specific points within the repeated DNA, possibly mediated by the sequence 5'-AAGG-3' 3'-TTCC-5' or its invert 5'-GGAA-3' 3'-CCTT-5'. (iii) There are alternating domains of conserved and divergent DNA sequences within each greater than 170-base-pair wing sequence. Taken together, the DNA sequences of these primates suggest a model whereby highly repeated DNAs are established and evolve as a consequence of unequal nonrandom exchanges of DNA duplexes. These exchanges may be mediated by short repeated nucleotide sequences and involve exchanges within and between the greater than 170-base-pair wings.

Animals↗

PubMed1985

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.

Hydrogen Bonding↗

PubMed2005

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition.

Spliceosome formation is initiated by the recognition of the 5' splice site through formation of an RNA duplex between the 5' splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5' splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5' splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5' splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5' splice site interaction does not decrease splicing efficiency, but rather increases 5' splice site recognition and exon inclusion. However, low complementarity of the 5' splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.

Base Pairing↗

PubMed1989

Theoretical prediction of base sequence effects in DNA. Experimental reactivity of Z-DNA and B-Z transition enthalpies.

Molecular modeling is used to study the sequence dependence of conformation and stability within helically regular duplex Z-DNA. The variations of conformation that are found are sufficiently important to be classified as a new type of polymorphism within the Z family. It is also demonstrated that certain sequences can adopt more than one of these polymorphic forms. Comparison with experimental studies of chemical reactivity within a natural DNA fragment, forced into a left-handed conformation, suggests that the results of our modeling may be used to explain the chemical reactivity observed. Comparison of the Z results with similar studies of the B form allow enthalpies of transition to be calculated as a function of base sequence.

Algorithms↗

PubMed2002

Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.

Base Sequence↗

PubMed1973

Self-complementarity of terminal sequences within plus or minus strands of adenovirus-associated virus DNA.

At least 70% of plus or minus strands of adenovirus-associated virus DNA contain self-complementary sequences at or near their termini. Self-annealing of these sequences generates circular molecules that are closed by duplex, hydrogen-bonded segments. The self-annealed segments are sensitive to exonuclease III and have a thermal stability comparable to that of double-stranded DNA molecules. Length measurements of double-stranded adenovirus-associated virus DNA molecules show a bimodal distribution, with the larger component being 10% shorter than SV40 DNA. The presence of self-complementary terminal sequences in single-stranded molecules of viral DNA has been observed previously only with DNA from adenoviruses. It is thus especially notable that adenovirus-associated virus replication is unconditionally dependent on a helper adenovirus. A possible role for terminal self-complementary sequences in viral DNA replication is suggested.

Adenoviridae↗

PubMed1992

Solution structure of [d(ATGAGCGAATA)]2. Adjacent G:A mismatches stabilized by cross-strand base-stacking and BII phosphate groups.

The solution structure of a rather unusual B-form duplex [d(ATGAGCGAATA)]2 has been determined using two-dimensional nuclear magnetic resonance (2D-NMR) and distance geometry methods. This sequence forms a stable ten base-pair B-form duplex with 3' overhangs and two pairs of adjacent G:A mismatches paired via a sheared hydrogen-bonding scheme. All non-exchangeable protons, including the stereo-specific H-5'S/H-5'R of the 3G and 7G residues, were assigned by 2D-NMR. The phosphorus spectrum was assigned using heteronuclear correlation with H-3' and H-4' reasonances. The complete assignments reveal several unusual nuclear Overhauser enhancements (NOEs) and unusual chemical shifts for the neighboring G:A mismatch pairs and their adjacent nucleotides. Inter-proton distances were derived from time-dependent NOEs and used to generate initial structures, which were further refined by iterative back-calculation of the two-dimensional nuclear Overhauser enhancement spectra; 22 final structures were calculated from the refined distance bounds. All these final structures exhibit fully wound helical structures with small penalty values against the refined distance bounds and small pair-wise root-mean-square deviation values (typically 0.5 A to 0.9 A). The two helical strands exchange base stacking at both of the two G:A mismatch sites, resulting in base stacking down each side rather than down each strand of the twisted duplex. Very large twist angles (77 degrees) were found at the G:A mismatch steps. All the final structures were found to have BII phosphate conformations at the adjacent G:A mismatch sites, consistent with observed downfield 31P chemical shifts and Monte-Carlo conformational search results. Our results support the hypothesis that 31P chemical shifts are related to backbone torsion angles. These BII phosphate conformations in the adjacent G:A mismatch step suggest that hydrogen bonding of the G:A pair G-NH2 to a nearby phosphate oxygen atom is unlikely. The unusual structure of the duplex may be stabilized by strong interstrand base stacking as well as intrastrand stacking, as indicated by excellent base overlap within the mismatch stacks.

Base Composition↗

PubMed1994

Can hammerhead ribozymes be efficient tools to inactivate gene function?

In order to improve hammerhead ribozyme efficiency and specificity, we have analyzed, both in vitro and in vivo, the activity of a series of ribozyme/substrate combinations that have the same target sequence but differ in the length of the ribozyme/substrate duplex or in their structure, i.e., the total length of the RNA. In vitro, we have found that optimal kcat/Km (at 37 degrees C) is obtained when the ribozyme/substrate duplex has a length of 12 bases, which according to the base composition represents a calculated free energy of binding of -16 kcal/mol. We discuss the importance of this value for ribozyme specificity and present strategies that may improve it. Increasing the length of the duplex from 14 to 17 bases (from -19 to -26 kcal/mol) produces a reduced ribozyme activity which is probably due to a slower rate of product dissociation. In addition, inclusion of either the substrate or the ribozyme in a long transcript produces a reduction (10 fold) of the kcat/Km, probably because of a different accessibility of the target sequence. In vivo, the activity of the trans-acting ribozyme was extremely low and detected in only one case: with a ribozyme/substrate duplex length of 13 bases and with both ribozyme and substrate embedded in short RNAs expressed at a very high level. The similarity of the results obtained in vitro and in vivo indicates that it is possible to use an in vitro system to optimize ribozymes which are to be used in vivo. Satisfactory results were obtained in vivo only with cisacting ribozymes. Altogether these results suggest that the ribozyme/substrate hybridization step is the limiting step in vivo and therefore it is not clear if ribozymes represent an improvement over antisense RNAs.

Base Sequence↗

PubMed2000

Proteins binding to duplexed RNA: one motif, multiple functions.

Highly structured and double-stranded (ds) RNAs are adaptable and potent biochemical entities. They interact with dsRNA-binding proteins (RBPs), the great majority of which contain a sequence called the dsRNA-binding motif (dsRBM). This approximately 70-amino-acid sequence motif forms a tertiary structure that interacts with dsRNA, with partially duplexed RNA and, in some cases, with RNA-DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or more dsRBMs. The motif also participates in complex formation through protein-protein interactions.

Amino Acid Motifs↗

PubMed1979

Precursor-specific nucleotide sequences can govern RNA folding.

An immediate precursor of 5S ribosomal RNA (rRNA) from Bacillus subtilis has 21 and 42 nucleotide precursor-specific segments associated with its 5' and 3' termini, respectively. On the basis of its nucleotide sequence, predicted secondary structure and location in the rRNA transcriptional unit, the 3' precursor element apparently functions during the termination of transcription. A portion of the 5' precursor element is shown to facilitate the native folding of the mature domain of the precursor. Precursor 5S rRNA molecules which lack the 5' terminal 8-9 nucleotides of the 5' precursor elements were fabricated. These abbreviated constructs assume a non-native conformation, as revealed by their behavior during polyacrylamide gel electrophoresis. The aberrant conformation is evidently forced upon the abbreviated constructs by the residual 5' precursor sequence, since its removal by the maturation endonuclease RNAase M5 precipitates the reordering of the mature domain into its native conformation. Inspection of the nucleotide sequence of the 5S precursor suggested the nature of the conformational aberration, and gel electrophoresis analyses of limited nuclease digests of end-labeled precursors in the native and aberrant conformations are consistent with the derived model. We conclude taht the 5' terminal six nucleotides in the intact 5S precursor assist in the folding of the mature domain by forming a base-paired duplex with neighboring nucleotides, thereby preventing that adjacent sequence from engendering the abnormal conformation. The involvement of precursor-specific sequences and conformational dynamics in RNA function are discussed.

Bacillus subtilis↗

PubMed1992

9-Aminoellipticine-derivatized alpha- and beta-oligodeoxyribonucleotides targeted to the cap of beta-globin mRNA: hybridization to natural and engineered mRNA, inhibition of translation, and improved effect of tandem chains.

We studied the duplex stability and the antimessenger activity of 9-aminoellipticine-5'-functionalized alpha- and beta-anomeric DNA sequences complementary to the first 14 nucleotides of the rabbit beta-globin mRNA. The duplex formed by the beta-conjugate with the natural mRNA target possessed a marginally better stability to that of the duplex formed by the unfunctionalized compound, as measured by the thermal elution. The alpha-conjugate did not anneal to native mRNA, possibly due to the interference of the 9-aminoellipticine with the cap structure and, unlike the beta-adduct, was practically inactive as inhibitor of translation in a cell-free system. However, it did hybridize to an RNA construction containing the beta-globin mRNA plus an additional 50 bases in 5'. Surprisingly, translation from this construction was inhibited by the alpha-species in spite of the nonvicinity of the target to the cap. Both alpha and beta conjugates hybridized to a DNA 14-mer of the same sequence as that targeted onto the mRNA. Thermal denaturation and fluorescence spectroscopy showed that the drug brought no considerable stabilization to the duplex, the linker presumably being unfavorable to intercalation. An increased stability of the complex and a higher inhibitory effect on cell-free beta-globin translation were obtained with two contiguous beta-oligomers of which one was functionalized.

Animals↗

PubMed1978

Isolation and characterization of a highly repetitious inverted terminal repeat sequence from Oxytricha macronuclear DNA.

The low-complexity "gene-sized" linear DNA duplexes of the Oxytricha macronucleus sport short inverted terminal repeats; thus, each single strand is capable of forming a circle held together by a duplex "neck" [Wesley, R. D. (1975) Porc. Natl. Acad. Sci. USA 72, 678--682]. We have isolated necks from total, circularized, single-stranded macronuclear DNA by treatment with nuclease S1. Necks represent at least 2.2% of the total DNA, are homogeneous in size (23 base pairs), melt at 55 degrees in 0.18 M Na+, and reassociate extremely rapidly at 22 degrees (Cot1/2 = 1.1 X 10(-5) mol-liter-1.sec) to form hybrid necks of the same thermal stability. From these and other results, we conclude that all necks on all the many thousands of different single-stranded circles are the same. The neck sequence is therefore highly repetitious--found in multiple copies (as inverted terminal repeats at flush duplex ends and probably also internally) on each natural "gene-sized" macronuclear DNA molecule--implying the possible participation of this sequence both in the general vegetative metabolism of macronuclear DNA and in the pre-vegetative process whereby macronuclear DNA is excised from the total Oxytricha genome.

Animals↗