RESUMEN
Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (micron ESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by micron ESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.
Asunto(s)
Oligonucleótidos/química , ARN/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Bases , Datos de Secuencia Molecular , Oligonucleótidos/síntesis químicaRESUMEN
The canonical double-helix form of DNA is thought to predominate both in dilute solution and in living cells. Sequence-dependent fluctuations in local DNA shape occur within the double helix. Besides these relatively modest variations in shape, more extreme and remarkable structures have been detected in which some bases become unpaired. Examples include unusual three-stranded structures such as H-DNA. Certain RNA and DNA strands can also fold onto themselves to form intrastrand triplexes. Although they have been extensively studied in vitro, it remains unknown whether nucleic acid triplexes play natural roles in cells. If natural nucleic acid triplexes were identified in cells, much could be learned by examining the formation, stabilization, and function of such structures. With these goals in mind, we adapted a pattern-recognition program to search genetic databases for a type of potential triplex structure whose presence in genomes has not been previously investigated. We term these sequences Potential Intrastrand Triplex (PIT) elements. The formation of an intrastrand triplex requires three consecutive sequence domains with appropriate symmetry along a single nucleic acid strand. It is remarkable that we discovered multiple copies of sequence elements with the potential to form one particular class of intrastrand triplexes in the fully sequenced genomes of several bacteria. We then focused on the characterization of the 25 copies of a particular approximately 37 nt PIT sequence detected in Escherichia coli. Through biochemical studies, we demonstrate that an isolated DNA strand from this family of E. coli PIT elements forms a stable intrastrand triplex at physiological temperature and pH in the presence of physiological concentrations of Mg(2+).
Asunto(s)
Biología Computacional/métodos , ADN/química , ADN/genética , Escherichia coli/genética , Genoma Bacteriano , Conformación de Ácido Nucleico , Algoritmos , Secuencia de Bases , Cromosomas Bacterianos/genética , ADN/clasificación , ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Bases de Datos Factuales , Genes Bacterianos/genética , Genómica/métodos , Calor , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Reconocimiento de Normas Patrones Automatizadas , Mapeo Físico de Cromosoma , Secuencias Reguladoras de Ácidos Nucleicos/genética , Alineación de Secuencia , Programas Informáticos , Espectrofotometría UltravioletaRESUMEN
Nucleic acid triple helices have provoked interest since their discovery more than 40 years ago, but it remains unknown whether such structures occur naturally in cells. To pursue this question, it is important to determine the stabilities of representative triple helices at physiological temperature and pH. Previous investigations have concluded that while both DNA and RNA can participate in the pyrimidine triplex motif under mildly acidic conditions, these structures are often relatively unstable at neutral pH. We are now explorin g the stability of intrastrand DNA and RNA pyrimidine motif triplexes at physiological temperature and pH. Duplex and triplex formation were monitored by thermal denaturation analysis, circular dichroism spectroscopy and gel shift experi-ments. Short intrastrand triplexes were observed to form in the pyrimidine motif in both DNA and RNA. In the presence of physiological concentrations of Mg(2+)and at physiological pH, all detected triplexes were sufficiently stable to persist at physiological temperature. If sequences specifying such intrastrand triplexes are encoded in genomes, the potential exists for the formation of stable structures in RNA or DNA in vivo.