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PubMed1994

A thermodynamic investigation of the melting of B-Z junction forming DNA oligomers.

Ultraviolet absorbance methods were used to characterize the thermodynamics of melting of a series of 16 bp deoxyoligonucleotides over a wide range of NaCl concentrations (0-4.5 M) and to obtain complete thermodynamic profiles for their melting at 0.115 and 4.5 M NaCl. The sequence of the series (one strand of duplex) was: 5'-CGCGCGCGAMNGACTG-3', where C indicates m5dC and -MN- was varied to include all combinations of Py:Py stacks (CC, TT, CT, TC). The unmethylated deoxyoligonucleotide 5'-CGCGCGCGACTGACTG-3' was used as a control sequence. All of the methylated oligonucleotides studied undergo a NaCl-induced transition to a hybrid form containing a left-handed, Z-DNA, region joined to a right-handed region by a B-Z junction. Our experiments allowed us to quantitatively evaluate the effects of NaCl, sequence, and methylation and the transition to the hybrid BZ structure on DNA thermal stability. We found that alteration of a single dinucleotide step has profound effects on the thermal stabilities of the 16 bp fragments studied. Methylation was found to destabilize the double helix, resulting in a decrease in Tm. Transition to the hybrid BZ structure, somewhat surprisingly, was found to only slightly destabilize DNA, with an observed decrease in free energy of melting of approximately 0.5 kcal/mol relative to the control, right-handed, sequence in high salt. Transition melting temperatures (Tm) were found, in agreement with previous studies on polymeric DNA, to depend upon NaCl concentration in a complicated, nonlinear fashion. m values increase to maximal values at circa 1.0 M NaCl, but decrease thereafter with further addition of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1989

Branch migration mediated DNA labeling and cloning.

The sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail can be mediated by branch migration into the end of a DNA molecule. Substitution of bromodeoxycytidine (BrdC) for deoxycytidine (dC) increased DNA-DNA hybrid stability. BrdC-containing oligodeoxynucleotides displaced dC-containing strands from duplexes with blunt ends or 3'-overhangs. In the later case the rate of displacement was of the same order of magnitude as DNA reassociation. A BrdC-containing displacer oligodeoxynucleotide was used for transient sequence-specific invasion at a particular PstI site. The product was captured by use of T4 DNA ligase and a linker oligodeoxynucleotide. The capture rate was more than 300 times the rate observed for an unrelated PstI site. This high degree of specificity required BrdC substitution. In addition, deliberate incorporation of an incorrect nucleotide into a displacer strand demonstrated that branch migration was terminated at a mismatch. A branched, BrdC-containing ligated product of a capture reaction was cloned and sequenced. The specific capture reaction may be used to label a particular DNA fragment prior to electrophoresis, to mark the specific fragment for affinity chromatography, or to facilitate cloning by introducing a new overhanging sequence compatible with a restriction endonuclease site in a cloning vector.

Autoradiography↗

PubMed2005

Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

Algorithms↗

PubMedDate not supplied

Peptide nucleic acids (PNA) and PNA-DNA chimeras: from high binding affinity towards biological function.

Oligonucleotide analogs are of major interest as tools in molecular biology, as diagnostics, and as potential pharmaceuticals which bind in a predictable way to certain nucleic acid target sequences, aiming at the inhibition of expression of disease-causing genes. One of the most promising nucleic acid mimetics are the peptide - or polyamide- nucleic acids (PNA) which bind with higher affinity to DNA and RNA than natural oligonucleotides. In these non-ionic PNAs, the entire sugar-phosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure. A unique property of PNA is its ability to displace one strand of a DNA double-helix. This strand displacement process, which is inefficient with DNA, is supported by the formation of an unusually stable internal (PNA)2 x DNA triple helix. The combination of PNA and DNA in one molecule results in PNA/DNA chimeras with new properties. They show improved aqueous solubility compared to pure PNAs due to their partially negatively charged structure. Furthermore, the cellular uptake of the chimeras is better than of pure PNAs. In contrast to PNA, the chimeras bind exclusively in the antiparallel orientation under physiological conditions. The binding affinity is generally stronger when the PNA/DNA chimeras are hybridized to RNAthan to DNA, whereby the strength of binding strongly depends on the PNA: DNA ratio. Most interestingly, PNA/DNA chimeras are recognized as substrates by various nucleic acid processing enzymes, and consequently can also assume biological functions, such as a primer function for DNA polymerases. Pure PNA cannot induce RNase H cleavage of target RNA, which is believed to support the biological efficacy of antisense agents. However, DNA-PNA chimeras are able to stimulate cleavage of the target RNA by RNase H upon formation of an RNA x chimera duplex.

Base Sequence↗

PubMed1994

The DNA cleavage pathway of iron bleomycin. Strand scission precedes deoxyribose 3-phosphate bond cleavage.

DNA strand scission initiated by bleomycin is a multistep process. Three C-C or C-O bonds are broken, releasing base propenal, a nucleic base derivative with deoxyribose carbons 1-3. Either C-3'-(phosphate-O) cleavage or C-3'-C-4' plus C-1'-(ring-O) bond cleavages could cause strand cleavage. To determine the sequence of bond breakage, d(CAAGCTTG) duplex was examined for rates of 1) strand scission, monitored by the hyperchromicity of cleavage-induced denaturation; 2) base propenal formation, monitored by 1H NMR spectroscopy; 3) 5'-terminal phosphomonoester formation, monitored by 31P NMR spectroscopy. Strand scission occurred with t 1/2 = 4.1 +/- 0.5 min at 4 degrees C, faster than base propenal formation (t 1/2 = 6.7 +/- 0.3 min). Thus newly cleaved DNA includes a base propenal precursor (t 1/2 = 2-3 min). The 5'-phosphate terminus forms (t 1/2 = 7.4 +/- 0.8 min) concurrently with base propenal. Since strand scission precedes phosphomonoester formation, strand scission cannot arise from C-3'-(phosphate-O) cleavage. Instead, the base propenal precursor must be linked to the future 5'-phosphate terminus, with strand scission arising from a combination of C-3'-C-4' and C-1'-(ring-O) bond cleavages. These results provide experimental support for a recently proposed mechanism that accommodates an early oxygen attack at C-4' and 2'-deprotonation without requiring simultaneous strand scission and 5'-phosphate terminus formation.

Base Sequence↗

PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗

PubMed1994

Structural characterization of PNA-DNA duplexes by NMR. Evidence for DNA in a B-like conformation.

The nucleic acid analogues PNA (peptide nucleic acids) hybridize with DNA of complementary sequence. The solution structures of two PNA-DNA duplexes, H-(GCTATGTC)-NH2.d(GACATAGC) and H-(GTAGATCACT)-NH2.d(AGTGATCTAC), have been studied by 1H NMR. It was found that the PNA-DNA hybrids are base paired by hydrogen bonds, most likely of the Watson-Crick type. From two-dimensional NOESY and COSY results it is concluded that the DNA strand in the PNA-DNA complex adopts a B-like structure with the deoxyribose sugars in the C2'-endo conformation.

Base Composition↗

PubMed2002

Studies on the mechanism of action of azinomycin B: definition of regioselectivity and sequence selectivity of DNA cross-link formation and clarification of the role of the naphthoate.

Evaluation of the sequence selectivity, noncovalent association, and orientation of the DNA cross-linking agent azinomycin B on its duplex DNA receptor is described. A strong correlation between sequence nucleophilicity and cross-linking yield was observed, and steric effects due to the thymine C5-methyl group were identified. Detailed studies on the role of the azinomycin naphthoate using viscometry, fluorescence contact energy transfer, and DNA unwinding assays point to a nonintercalative binding mode for this group. A kinetic assay for agent regioselectivity was used to determine the orientation of binding and covalent cross-link formation.

Anti-Bacterial Agents↗

PubMed2003

X-ray analyses of d(GCGAXAGC) containing G and T at X: the base-intercalated duplex is still stable even in point mutants at the fifth residue.

DNA fragments containing the sequence d(GCGAAAGC) prefer to adopt a base-intercalated (zipper-like) duplex in the crystalline state. To investigate effects of point mutation at the 5th residue on the structure, two crystal structures of d(GCGAGAGC) and d(GCGATAGC) have been determined by X-ray crystallography. In the respective crystals, the two octamers related by a crystallographic two-fold symmetry are aligned in an anti-parallel fashion and associated to each other to form a duplex, suggesting that the base-intercalated duplex is stable even when the 5th residue is mutated with other bases. The sheared G3:A6 pair formation makes the two phosphate backbones closer and facilitates formation of the A-X*-X-A* base-intercalated motif. The three duplexes are assembled around the three-fold axis, and their 3rd and 4th residues are bound to the hexamine cobalt chloride. The central 5th residues are bound to another cation.

Base Pairing↗

PubMed2002

Actinomycin D binds to d(TGTCATG) with 2:1 drug to duplex stoichiometry.

Despite the apparent single-stranded conformation and the absence of a GpC site, d(TGTCATTG) has been found to bind strongly to actinomycin D (ACTD) with 1:1 drug to strand binding stoichiometry. A hairpin binding model was speculated in which the planar phenoxazone chromophore inserts at the GTC site by pushing out the T-base while the terminal G folds back to form a G small middle dotC base pair so that the 3'-sides of both G-bases stack on the opposite faces of the phenoxazone plane. However, it was also suggested that a slipped duplex binding with similar binding principle could also be operative at higher DNA concentrations. To support such a contention, ACTD binding studies were made with d(TGTCATG) and related oligomers. This heptamer differs from the parent octamer d(TGTCATTG) by a mere removal of a T-base which should result in an enhancement of dimeric duplex formation and a concomitant reduction in monomeric hairpin contribution. It was found that ACTD binds well to d(TGTCATG) with 1 drug to 1 strand (or 2 drugs to 1 duplex) binding stoichiometry. These results are consistent with a slipped duplex binding model in which a dimeric duplex is formed at the self-complementary CATG tetranucleotide sequence with extruding TGT ends. Two drug molecules are bound at both ends of the duplex by pushing out the T-bases of GTC's so that the opposite faces of each phenoxazone are stacked by the 3'-sides of the two G-bases on opposite strands. Such a model provides a ready explanation for the observed enhancement in ACTD binding to d(TGTCATGTC) and d(TGTCATGTCA), where additional base pairs at the ends will stabilize GTC/GTC binding sites, and to d(TGTCAATTG) in which two additional base pairs facilitate the slipped-duplex formation. The observed ACTD affinity reductions for oligomers containing GTTC instead of GTC are also consistent with the T-base displacement model. These findings greatly expand the repertoire of ACTD binding to DNA and may have important implications on understanding the transcription inhibitory activities of this drug.

Base Sequence↗

PubMed1996

Improved nearest-neighbor parameters for predicting DNA duplex stability.

Thermodynamic data were determined from UV absorbance vs temperature profiles of 23 oligonucleotides. These data were combined with data from the literature for 21 sequences to derive improved parameters for the 10 Watson-Crick nearest neighbors. The observed trend in nearest-neighbor stabilities at 37 degrees C is GC > CG > GG > GA approximately GT approximately CA > CT > AA > AT > TA (where only the top strand is shown for each nearest neighbor). This trend suggests that both sequence and base composition are important determinants of DNA duplex stability. On average, the improved parameters predict deltaG degrees(37), deltaH degrees, deltaS degrees, and T(m) within 4%, 7%, 8%, and 2 degrees C, respectively. The parameters are optimized for the prediction of oligonucleotides dissolved in 1 M NaC1.

Base Sequence↗

PubMed1994

A molecular rheostat. Co-operative rev binding to stem I of the rev-response element modulates human immunodeficiency virus type-1 late gene expression.

The complete biologically active human immunodeficiency virus type-1 (HIV-1) rev-response element (RRE) RNA is 351 nucleotides (nt) in length, and includes an extra 58 nt on the 5' end and 59 nt on the 3' end beyond the sites proposed in the original models for the RRE secondary structure. The extra sequences are able to form a duplex structure which extends Stem I. The presence of an elongated Stem I structure in the RRE RNA was confirmed by nuclease mapping experiments. Nuclease protection experiments have shown that rev binds to restricted regions of the RRE, including the high affinity site located at the base of Stem IIb and along the length of the Stem I region. The three large stem-loop structures which protrude from Stem I and Stem IIb (Stems IIc, III+IV and V) remain accessible to nucleases even in the presence of a large excess of protein. Gel-retardation experiments show that the truncations of Stem I reduced the total number of rev molecules that can bind co-operatively and with high affinity to the RRE RNA. To test whether the elongated Stem I structure is required for maximal rev activity, a series of truncations which progressively reduced the length of Stem I was introduced into an HIV-1 derived reporter plasmid. In the presence of rev and a functional RRE, there is an increase in the levels of gag and env mRNA in the cytoplasm and a decrease in levels of tat and rev mRNAs. Each of the truncations in Stem I reduced the rev responses, with the longest truncations producing the greatest losses of activity. The data suggest that the RRE acts as a "molecular rheostat" designed to detect rev levels during the early stages of the HIV growth cycle.

Base Sequence↗

PubMed1992

Triple-helix formation and cooperative binding by oligodeoxynucleotides with a 3'-3' internucleotide junction.

Triple-helix formation by oligodeoxynucleotides in a sequence-specific manner is limited to polypurine tracts of duplex DNA. To increase the number of biologically relevant targets for triple-helix formation, we have utilized oligodeoxynucleotides containing a 3'-3' internucleotide junction to allow for binding to opposite strands of duplex DNA. Molecular modeling was used to aid in the design of the xylose dinucleoside linker 1 that is rigid and minimizes the number of conformers to minimize the entropy of binding. Thermal denaturation studies show that a 3'-3'-linked oligodeoxynucleotide, bearing nine nucleotides on each side of the linker, has a higher Tm (47.6 degrees C) than that of a 21-mer binding to a single polypurine tract (45.3 degrees C). Binding domain minimization studies and sequence-specific alkylation of a target duplex demonstrate a high degree of cooperativity between the two triple-helix binding domains, thus allowing for an increase in the number of biologically relevant targets for triple-helix formation.

Base Composition↗

PubMed1983

Proton NMR studies on the covalently linked RNA-DNA hybrid r(GCG)d(TATACGC). Assignment of proton resonances by application of the nuclear Overhauser effect.

Proton NMR spectra of a covalently linked self-complementary RNA X DNA hybrid, r(GCG)-d(TATACGC), are recorded in H2O and D2O. Imino proton resonances as well as the non-exchangeable base and H-1' resonances are unambiguously assigned by means of nuclear. Overhauser effect measurements. Additional information was obtained by 31P NMR and circular dichroism spectra. The RNA parts in the duplex attain full conformational purity and adopt the usual A-RNA conformation. The DNA residues opposite the RNA tract do not adopt an A-type structure completely. Their respective sugar rings still appear to possess a certain conformational freedom. The same holds true for the central d(-TATA-) sequence which forms a DNA X DNA duplex. There appears to be a structural break in this part: the first two residues, T(4) and A(5), are clearly influenced by the adjacent RNA structure, whereas residues T(6) and A(7) behave quite similar to what usually is found in DNA duplexes in aqueous solution.

Base Sequence↗

PubMed2002

DNA recognition control of gamma-PRNA and mismatched base effects upon complex stability.

Oligomeric peptide ribonucleic acids (PRNA's), tethering 5'-amino-5'-deoxyribonucleosides as a recognition site for nucleic acids, has been designed and synthesized by solid phase method. PRNA 12-mer found to form stable duplex with complementary DNA.PRNA.DNA duplex composed of PRNA with mismatched DNA has 10-degree lower melting temperature than fullmatched PRNA.DNA duplex. These results demonstrate that binding of PRNA with DNA is sequence specific. It should be emphasized that the on-off switching of PRNA.DNA duplex formation has been realized by the borate added as an external factor.

Base Pair Mismatch↗

PubMed1993

Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.

RNAs modified with the chemical nuclease 1,10-phenanthroline-copper(I) can achieve the sequence-specific scission of single- and double-stranded DNA targets. The RNAs are prepared in vitro by using 5-(3-aminoallyl)-UTP as the sole source of UTP and can be readily modified with 1,10-phenanthroline by using N-succinimidyl 3-(2-pyridyl-dithio)propionate (SPDP) to cross-link the ligand to the aminoallyl moiety. Single-stranded DNAs are efficiently cleaved at multiple sites because 1,10-phenanthroline is incorporated at several uridines in the sequence. Sequence-specific double-stranded scission of duplex DNA can also be accomplished with 1,10-phenanthroline-derivatized RNA within R loops. These triple-stranded structures form in 70% formamide and involve the displacement of one strand of DNA by the RNA of identical sequence. R loop-directed scission is the first method for DNA scission applicable to any sequence. A unique application of R loop-targeted nucleolytic scission, which relies on its ability to cut DNA at any sequence, is the determination of the distance between two marker DNA sequences within a target. In this case, 1,10-phenanthroline-linked RNAs are prepared from the two distinct sequences and used to cut the DNA fragment after R-loop formation. The size of the fragment liberated by these methods is a direct measure in base pairs of the distance between the two DNA sequences. For example, the distance separating two chicken delta crystallin (delta 1 and delta 2) genes has been confirmed as 24 kilobases by this method.

Animals↗

PubMed1980

An abundant cytoplasmic 7S RNA is complementary to the dominant interspersed middle repetitive DNA sequence family in the human genome.

Evidence is presented that a homogeneous cytoplasmic species known as 7S RNA is the only abundant RNA in uninfected HeLa cells which can form strong hybrids with the dominant family of middle repetitive DNA sequences in the human genome. These DNA sequences are known collectively as the Alu family, because most of them share a common Alu I restriction site. When purified 7S RNA was hybridized to three different genomic clones containing Alu family DNA sequences, a specific region (or regions) comprising at most half the RNA sequence was protected from mild digestion with T1 ribonuclease; moreover, the hybrids between 7S RNA and cloned Alu family DNA wer imperfect, since T1 RNAase was able to nick the protected 7S RNA sequences under conditions where a true RNA: DNA duplex would have been resistant. This suggests that 7S RNA is encoded either by a small subset of the 300,000 Alu family sequences in the human genome or by an entirely different family of genes. The sequence of 7S RNA has been highly conserved through recent evolution, and in both avian and murine cells the RNA is selectively incorporated into oncornavirus particles during productive infection. The cellular function of 7S RNA is unknown.

Cytoplasm↗

PubMed2001

A 2'-acylamido cap that increases the stability of oligonucleotide duplexes.

[structure: see text]. Reported here is the synthesis of oligonucleotides containing a 2'-acylamido-2'-deoxyuridine residue at their 3'-terminus. Compared to control sequences bearing a thymidine residue, the quinolone-capped duplexes give higher UV melting points. In the case of (5'-ACGCGU-NA-2')2, where NA denotes a nalidixic acid residue, the melting point increase is up to 22 degrees C over that of (ACGCGT)2.

Anti-Infective Agents↗