ABSTRACT
We have synthesized diastereomerically pure diadenosine 3',5'-boranophosphates (Ap(b)A) by using the boranophosphotriester method from ribonucleosides protected with the 2'-hydroxy protecting group 2-cyanoethoxymethyl (CEM). Melting curves of the triple-helical complex of the dimer Ap(b)A and 2poly(U) at high ionic strength revealed that presumptive (Sp)-Ap(b)A had a much higher affinity and presumptive (Rp)-Ap(b)A a much lower affinity for poly(U) than the natural dimer ApA did. In contrast, the affinities of these dimers for poly(dT) were similar. Both the (Rp)- and the (Sp)-boranophosphate diastereomers showed much higher resistance to digestion by snake venom phosphodiesterase and nuclease P1 than ApA did. They have potential for use as synthons to be incorporated into boranophosphate oligonucleotides. In particular, because oligonucleotides containing Sp boranophosphate nucleotides are expected to bind more strongly and specifically to RNA than natural oligoribonucleotides do, they may find application in the isolation and detection of functional RNA in basic research and diagnostics.
Subject(s)
Adenosine/chemistry , Boranes/chemical synthesis , Cyanides/chemistry , Ethyl Ethers/chemistry , Phosphates/chemical synthesis , Boranes/chemistry , Boranes/metabolism , Esterases/metabolism , Magnetic Resonance Spectroscopy , Methylation , Molecular Structure , Nucleic Acid Denaturation , Phosphates/chemistry , Phosphates/metabolism , Stereoisomerism , Substrate SpecificityABSTRACT
A novel method for the synthesis of RNA oligomers with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group has been developed. The new method allows the synthesis of oligonucleotides with an efficiency and final purity comparable to that obtained in DNA synthesis.(1) In addition, the CEM method has the potential for application to the synthesis of very long RNA oligonucleotides.
Subject(s)
Ethyl Ethers/chemistry , Methyl Ethers/chemistry , Oligoribonucleotides/chemical synthesis , RNA/chemical synthesis , Oligoribonucleotides/chemistry , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , RNA/chemistrySubject(s)
Drosophila Proteins/chemistry , Histone Acetyltransferases/chemistry , TATA-Box Binding Protein/chemistry , Transcription Factor TFIID/chemistry , Magnetic Resonance Spectroscopy , Protein Conformation , TATA Box , TATA-Binding Protein Associated Factors , TATA-Box Binding Protein/metabolism , Transcription Factor TFIID/metabolism , Transcription, GeneticABSTRACT
A novel method for the synthesis of RNA oligomers with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group has been developed. The new method allows the synthesis of oligoribonucleotides with an efficiency and final purity comparable to that obtained in DNA synthesis. [structure: see text]
Subject(s)
Nitriles/chemistry , RNA/chemical synthesis , Base Sequence , Models, Chemical , Molecular Sequence Data , Molecular Structure , RNA/chemistryABSTRACT
Oligonucleotides containing a methylene bridge between N1 or N9 of the heterocyclic base and C1' of the pentofuranosyl ring (homo-N-oligonucleotides) were synthesized. Melting curves revealed that such homo-type oligomers could cross-pair with complementary homo-type or natural oligomers. Circular-dichroic studies provide evidence that the homo-type dimers have a left-handed stacked conformation and further suggest that single-stranded and double-stranded homo-type oligomers adopt a left-handed conformation, while duplexes with natural oligomers or nucleic acids form RNA-like right-handed helices. NMR spectroscopy (NOESY) provides supporting evidence for a left-handed stacked conformation of the homo-type dimer, while atomic force microscopy indicates a left-handed helical conformation of homo-type dsDNA. Homo-type dimers and oligomers showed high resistance to digestion by snake-venom and calf-spleen phosphodiesterases and nuclease S1.
Subject(s)
Nucleic Acids/chemistry , Oligonucleotides/chemistry , Oligonucleotides/chemical synthesis , Circular Dichroism , Microscopy, Atomic Force , Models, Molecular , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Nucleic Acids/metabolism , Oligonucleotides/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively). Our NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region.
Subject(s)
Saccharomyces cerevisiae/metabolism , TATA-Box Binding Protein/metabolism , Transcription Factor TFIID/metabolism , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Polymerase Chain Reaction , Protein Conformation , Protein Folding , Sequence Homology, Amino Acid , TATA-Box Binding Protein/chemistry , Transcription Factor TFIID/chemistry , Transcription Factor TFIID/genetics , Two-Hybrid System TechniquesABSTRACT
Oligonucleotides containing a methylene bridge between Nl or N9 of the heterocyclic base and C1' of the pentofuranosyl ring (homo-N-oligonucleotides) were synthesized. Melting curves revealed that such homo-type oligomers could cross-pair with complementary homo-type or natural oligomers. Circular dichroism spectra provide evidence that the homo-type dimers have a left-handed stacked conformation and further suggest that single-stranded and double-stranded homo-type oligomers adopt a left-handed conformation, while duplexes with natural oligomers or nucleic acids form right-handed helices. NMR (NOESY) provides supporting evidence for a left-handed conformation of the homo-type dimer. Atomic force microscopy (AFM) indicates a left-handed helical conformation of homo-type dsDNA.
