RESUMEN
A 2'-O-methyl-RNA oligonucleotide containing a single free 2'-OH group flanking a branching phosphotriester linkage was prepared as a model for phosphate-branched RNA by using an orthogonally protected dimeric phosphoramidite building block in solid-phase synthesis. The strategy allows the synthesis of phosphate-branched oligonucleotides, the three branches of which may be of any desired sequence. Hydrolytic reactions of the phosphotriester linkages in such oligonucleotides were studied at physiological pH in the presence (and absence) of various complementary oligonucleotides. The fully hybridized oligonucleotide model is an order of magnitude more stable than its single-stranded counterpart, which, in turn, is an order of magnitude more stable than its trinucleoside phosphotriester core lacking any oligonucleotide arms. Furthermore, kinked structures obtained by hybridizing the phosphate-branched oligonucleotide with partially complementary oligonucleotides are three to five times more stable than fully double-stranded ones and only approximately three times less stable than the so-called RNA X structure, which has been postulated to incorporate an RNA phosphotriester linkage. The results indicate that when the intrinsically unstable RNA phosphotriester linkage is embedded in an oligonucleotide of appropriate tertiary structure, its half-life can be at least several hours.
Asunto(s)
Fosfatos/química , Estabilidad del ARN , ARN/química , Secuencia de Bases , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Motivos de Nucleótidos , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Compuestos Organofosforados/química , ARN/genética , ARN/metabolismoRESUMEN
A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereoselectivity (≥98:2). The coupling efficiency was greatly improved by the use of 2-cyanoethoxymethyl (CEM) groups in place of the conventional TBS groups for the 2'-O-protection of nucleoside 3'-O-oxazaphospholidine monomers. The resultant diastereopure PS-ORNs allowed us to clearly demonstrate that an ORN containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a destabilizing effect.
Asunto(s)
Nucleósidos/química , Oligorribonucleótidos/síntesis química , Oxazoles/química , Fosfatos/síntesis química , Tionucleótidos/síntesis química , Sustancias Macromoleculares , Oligorribonucleótidos/química , Fosfatos/química , Técnicas de Síntesis en Fase Sólida , Estereoisomerismo , Tionucleótidos/químicaRESUMEN
Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.
Asunto(s)
VIH-1/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Proteínas de la Nucleocápside/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/biosíntesis , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismoRESUMEN
{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4'-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2'-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2'-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn(2+). Introduction of Zn(2+) moderately stabilized the duplex with U-bulged targets.
Asunto(s)
Complejos de Coordinación/química , Compuestos Heterocíclicos/química , Oligorribonucleótidos/química , Zinc/química , Ciclamas , Uracilo/químicaRESUMEN
In this paper, we present a method for the sensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes and a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22-mer oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 amol). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis as well as a range of other RNA based detection applications.
Asunto(s)
Anticuerpos/inmunología , ADN/química , MicroARNs/análisis , Hibridación de Ácido Nucleico/métodos , ARN/química , Silicio/química , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN/inmunología , Inmunoensayo/métodos , Ratones , Oligorribonucleótidos/química , ARN/inmunologíaRESUMEN
2'-O,4'-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA-DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg(2+)- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.
Asunto(s)
ADN/química , Magnesio/química , Ácidos Nucleicos Heterodúplex/química , Oligonucleótidos/química , Oligorribonucleótidos/química , ARN/química , Cinética , TermodinámicaRESUMEN
We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.
Asunto(s)
Oligorribonucleótidos/química , Cromatografía Líquida de Alta Presión , Ciclotrones , Análisis de Fourier , Espectrometría de Masas , Fosfatos/químicaRESUMEN
The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).
Asunto(s)
Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/síntesis química , Evolución Molecular Dirigida , Oligorribonucleótidos/química , Tionucleótidos/síntesis química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntesis química , Secuencia de Bases , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , ADN Complementario/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Oligorribonucleótidos/metabolismo , Ribonucleasa Pancreática/metabolismo , Tionucleótidos/química , Tionucleótidos/metabolismo , Trombina/metabolismo , Transcripción Genética , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virales/metabolismoRESUMEN
2'-O-Methyl oligoribonucleotides bearing a 3'-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn(2+) ions complementary oligoribonucleotides at the 5'-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn(2+) 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2'-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-beta-d-erythro-pentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn(2+) with Cu(2+) or Ni(2+) retards the cleaving activity of all the cleaving agents tested.
Asunto(s)
Compuestos Aza/metabolismo , Ciclodecanos/metabolismo , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Zinc/química , Secuencia de Bases , Catálisis , Hidrólisis , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Zinc/metabolismoRESUMEN
Right-handed RNA duplexes of (CG)n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)2 determined in 6 M NaClO4. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6(n)-H5'/H5''(n)-H8(n+1)-H1'(n+1)-H6(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4-7 degrees), whereas GpC steps have large negative values (-61 degrees). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2'-OH groups, and the major groove are well defined. The 2'-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2'-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.
Asunto(s)
Modelos Moleculares , Oligorribonucleótidos/química , ARN Bicatenario/química , Emparejamiento Base , Secuencia de Bases , Citidina/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Fósforo/química , Protones , Cloruro de Sodio/químicaRESUMEN
Nuclear factor-kappaB (NF-kappaB) transcription factors are important in regulating the immune response and play critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. Agents that target specific NF-kappaB dimers may serve as therapeutic agents for the prevention of pathogenic immune responses. We have selected monothiophosphate-modified aptamers, or "thioaptamers", to the NF-kappaB p50/RelA heterodimer using combinatorial selection techniques. We also utilized a "double sieve" or editing approach for the generation of thioaptamers with enhanced selectivity to the RelA/RelA homodimer. The thioaptamers from these selections and our previous selections on the p50/p50 and RelA/RelA homodimers all had unique sequences and bound tightly to the recombinant NF-kappaB dimers against which they were selected. The selected thioaptamers also appear to maintain their selectivity and specificity among other cellular proteins, because they have the ability to bind NF-kappaB proteins within nuclear extracts from lipopolysaccharide (LPS)-induced macrophages and B cells.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , FN-kappa B/metabolismo , Oligorribonucleótidos/farmacología , Fosfatos , Linfocitos B/química , Secuencia de Bases , Núcleo Celular/química , Dimerización , Humanos , Lipopolisacáridos , Macrófagos/química , FN-kappa B/efectos de los fármacos , Subunidad p50 de NF-kappa B , Oligorribonucleótidos/química , Factor de Transcripción ReIA , Factores de TranscripciónRESUMEN
We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2'-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2'-methylseleno cytidine phosphoramidite and improved the accessibility of the 2'-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2'-selenium derivatization had no effect on the stability of the duplexes with the 3'-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3'-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2'-Se-C and 2'-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2'-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.
Asunto(s)
Cristalografía por Rayos X , Cisteína/análogos & derivados , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/química , Selenio/química , Secuencia de Bases , Cristalización , Cisteína/química , ADN/química , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligorribonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Compuestos de Organoselenio/química , ARN/química , Selenocisteína/análogos & derivados , Uridina/análogos & derivadosRESUMEN
The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.
Asunto(s)
Virus de la Hepatitis Delta/fisiología , ARN Catalítico/química , Secuencia de Bases , Calcio/farmacología , Genoma Viral , Virus de la Hepatitis Delta/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Manganeso/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Fosforilación , ARN Catalítico/metabolismo , Especificidad por Sustrato , Termodinámica , Replicación ViralRESUMEN
We have demonstrated that oligoribonucleotides that lack a 3'-OH group and cannot be extended by RNA polymerase can hybridize to the single-stranded DNA formed inside the transcription initiation bubble (or open complex) and inhibit transcription. Using the lacUV5/Escherichia coli RNA polymerase or trpEDCBA/E. coli RNA polymerase transcription system as a model, we have found that effective inhibitors are five nucleotides in length and must be complementary to the DNA template strand in the region from -5 to +2 about the transcription start site (designated +1). We have used the DNA cleavage activity of 1,10-phenanthroline-copper to confirm that the mechanism of inhibition is via oligoribonucleotide hybridization to the open complex and have used this cleavage chemistry to demonstrate that these oligonucleotide inhibitors hybridize in an antiparallel orientation to their DNA target. Systematic modification of the parent phosphodiester oligoribonucleotide pentamer revealed that the phosphorothioate backbone-containing analogs have increased open complex binding affinity and are more effective transcription inhibitors than their phosphodiester counterparts.
Asunto(s)
ADN/efectos de los fármacos , Técnicas Genéticas , Oligorribonucleótidos/química , Transcripción Genética/efectos de los fármacos , Secuencia de Bases , Cobre/química , ADN Complementario/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Modelos Químicos , Datos de Secuencia Molecular , Ácidos Nucleicos/química , Fenantrolinas/química , Regiones Promotoras GenéticasRESUMEN
Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
Asunto(s)
Endorribonucleasas/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Western Blotting , Regulación hacia Abajo , Activación Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Fosfatidiletanolaminas/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Procesamiento Postranscripcional del ARN/genética , ARN sin Sentido/química , ARN sin Sentido/genética , ARN sin Sentido/fisiología , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa P , Especificidad por Sustrato , Transfección/métodos , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Proteína bcl-X , ARN Pequeño no TraducidoRESUMEN
To construct a DNA-linked RNase H, which cleaves RNA site-specifically at high temperatures, the 15-mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA (PPT-RNA), was cross-linked to the unique thiol group of Cys135 in the Thermus thermophilus RNase HI variant. The resultant DNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH, in which the RNase H portion of the d15-C135/TRNH is replaced by the Escherichia coli RNase HI variant, cleaved the 15-mer PPT-RNA site-specifically. The mixture of the unmodified enzyme and the unlinked 15-mer DNA also cleaved the PPT-RNA but in a less strict manner. In addition, this mixture cleaved the PPT-RNA much less effectively than the DNA-linked enzyme. These results indicate that the cross-linking limits but accelerates the interaction between the enzyme and the DNA/RNA substrate. The d15-C135/TRNH cleaved the PPT-RNA more effectively than the d15-C135/ERNH at temperatures higher than 50 degrees C. The d15-C135/TRNH showed the highest activity at 65 degrees C, at which the d15-C135/ERNH showed little activity. Such a thermostable DNA-linked RNase H may be useful to cleave RNA molecules with highly ordered structures in a sequence-specific manner.
Asunto(s)
ADN/química , ADN/metabolismo , Oligodesoxirribonucleótidos/metabolismo , ARN Viral/metabolismo , ARN/metabolismo , Ribonucleasa H/metabolismo , Thermus thermophilus/enzimología , Sitios de Unión , Cisteína/química , Cartilla de ADN/química , ADN Viral/biosíntesis , VIH-1/enzimología , Calor , Cinética , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Purinas/química , Relación Estructura-ActividadRESUMEN
We selected RNA aptamers specific for the trans-activation responsive (TAR) RNA, a stem-loop structure crucial for the transcription of the integrated genome of the human immunodeficiency virus. Most of the selected sequences could be folded as imperfect hairpins and displayed a 5'-GUCCCAGA-3' consensus motif constituting the apical loop. The six central bases of this consensus sequence are complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer "kissing" complexes. The consensus G and A residues closing the aptamer loop contributed to the high affinity (Kd = 30 nM at 23 degrees C) of the aptamers for the TAR RNA. This G A pair was shown to be crucial for binding to TAR at a low magnesium concentration. The selection also identified 5'-PuPy and 5'-PyPu base pairs at alpha and beta positions of the stem, next to the loop, respectively. This strategy offered a way to identify key determinants of loop-loop interactions and to generate high affinity ligands of TAR RNA structure.
Asunto(s)
Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , ARN Viral/química , ARN Viral/genética , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Cartilla de ADN , Humanos , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Relación Estructura-ActividadRESUMEN
Until recently, there were no examples of RNAs whose structures had been determined by both NMR and x-ray crystallography, and thus there was no experimental basis for assessing the accuracy of RNA solution structures. A comparison of the solution and the crystal structures of two RNAs is presented, which demonstrates that NMR can produce solution structures that resemble crystal structures and thus validates the application to RNA of a methodology developed initially for the determination of protein conformations. Models for RNA solution structures are appreciably affected by the parameters used for their refinement that describe intramolecular interactions. For the RNAs of interest here, the more realistic those parameters, the greater the similarity between solution structures and crystal structures.
Asunto(s)
Oligorribonucleótidos/química , ARN/química , Secuencia de Bases , Fenómenos Biofísicos , Biofisica , Simulación por Computador , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Fósforo/química , Soluciones , Electricidad Estática , TermodinámicaRESUMEN
The crystal structure of the RNA octamer 5'-CGC(CA)GCG-3' has been determined from X-ray diffraction data to 2.3 A resolution. In the crystal, this oligomer forms a self-complementary double helix in the asymmetric unit. Tandem non-Watson-Crick C-A and A-C base pairs comprise an internal loop in the middle of the duplex, which is incorporated with little distortion of the A-form double helix. From the geometry of the C-A base pairs, it is inferred that the adenosine imino group is protonated and donates a hydrogen bond to the carbonyl group of the cytosine. The wobble geometry of the C-A+ base pairs is very similar to that of the common U-G non-Watson-Crick pair.
Asunto(s)
Adenina/química , Citosina/química , Conformación de Ácido Nucleico , ARN/química , Composición de Base , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Oligorribonucleótidos/química , TermodinámicaRESUMEN
Helix formation between various combinations of 3'-5' linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0-50 degrees C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)2, or triadenylic acid, (pA)3, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid. (pU)2, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)3, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, A --> U and U --> A, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2'-5' linked decauridylate, (pU)10, and pA or 2'-5' linked (pA)2 at 0 degrees C, which indicates the possibility of the template activity of long 2'-5' linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.