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1.
Aptamers targeting NF-kappaB containing thymidine 3'-O-phosphorodithioates in selected positions of an oligonucleotide duplex were synthesized. Binding affinities to NF-kappaB varied with the number and positions of the dithioate backbone substitutions. One of the aptamers showed specific binding to a single NF-kappaB dimer in cell culture extracts.  相似文献   

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Abstract

Assignment of the 1H and 31P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS? 2AAGCG)2 (10-mer-S; a site of dithioate substitution is designated with the symbols pS? 2), was achieved by two-dimensional homonuclear TOCSY, NOES Y and 1H-31P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCI), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 Å which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)2 (4.2~9.4Å). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.  相似文献   

4.
BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.  相似文献   

5.
The glycine binding riboswitch forms a unique tandem aptamer structure that binds glycine cooperatively. We employed nucleotide analog interference mapping (NAIM) and mutagenesis to explore the chemical basis of glycine riboswitch cooperativity. Based on the interference pattern, at least two sites appear to facilitate cooperative tertiary interactions, namely, the minor groove of the P1 helix from aptamer 1 and the major groove of the P3a helix from both aptamers. Mutation of these residues altered both the cooperativity and binding affinity of the riboswitch. The data support a model in which the P1 helix of the first aptamer participates in a tertiary interaction important for cooperativity, while nucleotides in the P1 helix of the second aptamer interface with the expression platform. These data have direct analogy to well-characterized mutations in hemoglobin, which provides a framework for considering cooperativity in this RNA-based system.  相似文献   

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Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.  相似文献   

8.
In vivo recognition of an RNA aptamer by its transcription factor target   总被引:2,自引:0,他引:2  
Cassiday LA  Maher LJ 《Biochemistry》2001,40(8):2433-2438
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Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this paper, new DNA binding molecules; diamino-bistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF ligand with RR configuration at the amino groups and C8 alkyl chains (RR8) stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined by isothermal titration calorimetry to be 3.3 x 10(8) M-1.  相似文献   

11.
Alternative models have been presented to provide explanations for the sequence-dependent variation of the DNA minor groove width. In a structural model groove narrowing in A-tracts results from direct, short-range interactions among DNA bases. In an electrostatic model, the narrow minor groove of A-tracts is proposed to respond to sequence-dependent localization of water and cations. Molecular dynamics simulations on partially methylphosphonate substituted helical chains of d(TATAGGCCTATA) and d(CGCGAATTCGCG) duplexes have been carried out to help evaluate the effects of neutralizing DNA phosphate groups on the minor groove width. The results show that the time-average minor groove width of the GGCC duplex becomes significantly more narrow on neutralizing the phosphate backbone with methylphosphonates. The minor groove of the AATT sequence is normally narrow and the methylphosphonate substitutions have a smaller but measurable affect on this sequence. These results and models provide a system that can be tested by experiment and they support the hypothesis that the electrostatic environment around the minor groove affects the groove width in a sequence-dependent dynamic and time-average manner.  相似文献   

12.
Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this paper, new DNA binding molecules; diamino-bistetrahydrofuran (bisTHF) and diamino-bisfuran are reported. The bisTHF ligand with RR configuration at the amino groups and C8 alkyl chains (RR8) stabilized GC-rich duplex. In contrast, bisfuran compounds stabilized AT-rich duplex. The binding affinity of RR8 with 12 mer duplex DNA was determined by isothermal titration calorimetry to be 3.3 × 108 M?1.  相似文献   

13.
In this work, we have developed a simple and sensitive method for ATP detection using silica nanoparticles (NPs) as the platform and hoechst33258 as the signal reporter. The ATP-binding aptamers hybridize with the probe DNA (DNA(p)) immobilized NPs to form the aptamer/DNA(p) duplex on the NPs surface. The conformational change of the aptamer leads to the decrease of the aptamer/DNA(p) duplex on the NPs due to the ATP-binding aptamer switches its structure from the aptamer/DNA(p) duplex to the aptamer/target complex in the presence of ATP. ATP detection can be easily realized by separating the silica nanoparticles and adding the hoechst33258 of intercalating to aptamer/DNA(p) (dsDNA). Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between ATP aptamer and ATP. The K(d) was estimated to be ~1mM from 0 to 4mM and a liner response was observed from 0 to 0.2mM with a detection limit of ~20μM. Compared with other methods, the carboxyl-modified silica nanoparticles (~60nm) prepared by the reverse microemulsion method can serve as a stable and sensitive sensor platform because of their smaller size and facile conjugation with amine-containing molecules. In addition, the high sensitivity and selectivity of hoechst33258 was employed for the ssDNA and dsDNA determination, which takes advantage of the label-free aptamer and lower cost.  相似文献   

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15.
To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.  相似文献   

16.
Interactions of DNA binding ligands with PNA-DNA hybrids.   总被引:1,自引:1,他引:0       下载免费PDF全文
The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.  相似文献   

17.
Side-by-side development of two competing technologies for obtaining affinity antibody-based and aptamer-based molecules opens new horizons for the creation of diagnostic and therapeutic agents of extremely high efficiency. Benefits of aptamers, such as relatively small size and selection simplicity, have been jeopardized for a long time by their intrinsic downsides, i.e., obscure process of obtaining aptamers against certain targets because of a low diversity of functional groups (purine and pyrimidine bases) in DNA and RNA aptamers. Another side effect of the aptamer technique inherent to the traditional SELEX method is unspecific enrichment with aptamers with high affinity to off-target reaction components. Today, due to current progress in the development of new technology methods and chemical coupling reactions, the modern aptamer technology helps to avoid its disadvantages and become capable of being the source of new diagnostic and therapeutic tools, which are properly unique in their efficiency. The review focuses on modern methods of increasing efficiency of the aptamer selection and on synthetic nucleotide modifications, which make it possible to prepare high-affinity aptamers against traditionally ‘hard’ targets.  相似文献   

18.
The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.  相似文献   

19.
Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.  相似文献   

20.
A phosphorothioate single-stranded DNA aptamer (thioaptamer) targeting transforming growth factor-beta1 (TGF-beta1) was isolated by in-vitro combinatorial selection. The aptamer selection procedure was designed to modify the backbone of single-stranded DNA aptamers, where 5' of both A and C are phosphorothioates, since this provides enhanced nuclease resistance as well as higher affinity than that of a phosphate counterpart. The thioaptamer selected from a combinatorial library (5x10(14) sequences) binds to TGF-beta1 protein with an affinity of 90 nM. In this report, sequence, predicted secondary structure, and binding affinity of the selected thioaptamer (T18_1_3) are presented.  相似文献   

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