ABSTRACT
We present a protocol for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. The approach is exemplified by tRNA(Val)-3'-NH-VFLVM-NH(2) and relies on commercially available Escherichia coli tRNA(Val). This tRNA was cleaved site-specifically within the TΨC loop using a 10-23 type DNA enzyme to obtain a 58 nt tRNA 5'-fragment which contained the modifications. After cleavage of the 2',3'-cyclophosphate moiety from the 5'-fragment, it was ligated to the 18 nt RNA-pentapeptide conjugate which had been chemically synthesized. By this methodology, tRNA(Val)-3'-NH-VFLVM-NH(2) is accessible in efficient manner. Furthermore, we point out that the approach is applicable to other types of tRNA.
Subject(s)
DNA, Catalytic/metabolism , Drug Resistance, Bacterial , Macrolides/pharmacology , Peptides , RNA Stability , RNA, Transfer, Amino Acyl/chemical synthesis , RNA, Transfer, Val/chemistry , Anti-Bacterial Agents/pharmacology , Base Sequence , Escherichia coli , Mass Spectrometry , Models, Molecular , Nucleic Acid Conformation , Phenol/chemistry , Phosphorylation , RNA, Bacterial/metabolism , RNA, Transfer, Val/chemical synthesis , RNA, Transfer, Val/isolation & purification , RNA, Transfer, Val/metabolismABSTRACT
The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.
Subject(s)
Peptides/chemistry , RNA, Transfer/chemistry , Base Sequence , DNA, Catalytic/metabolism , Hydrolysis , Molecular Sequence Data , RNA Ligase (ATP)/metabolism , RNA, Transfer/metabolismABSTRACT
Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.
Subject(s)
Molecular Mimicry , Peptides/chemistry , RNA, Transfer, Amino Acyl/chemical synthesis , RNA/chemistry , Amino Acid Sequence , Base Sequence , Hydrolysis , Protein Biosynthesis , RNA, Transfer, Amino Acyl/chemistryABSTRACT
Labeling of RNA with site-specific fluorine atoms has become straightforward in recent years and in particular has become an integrated part of engineered functional RNA with therapeutic potential, e.g. for siRNA technologies. Here, we demonstrate that temperature-dependent one-dimensional (19)F NMR spectroscopy of oligoribonucleotides is a powerful tool to obtain direct information on the conformational behavior. The approach is particularly useful for the quantification of monomolecular vs bimolecular secondary structure equilibria.
Subject(s)
Fluorine Radioisotopes/chemistry , Magnetic Resonance Spectroscopy/methods , RNA/chemistry , Hydrogen-Ion Concentration , Models, Chemical , Nucleic Acid Conformation , Nucleic Acids/chemistry , Oligoribonucleotides/chemistry , Protein Structure, Secondary , RNA, Small Interfering/metabolism , Temperature , Thermodynamics , Ultraviolet RaysABSTRACT
Site-specifically modified 2'-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels-Alder ribozyme,(1) have relied on these modifications.(2) Currently, preparation of 2'-methylseleno modified oligoribonucleotides is restricted to the 2'-O-[(triisopropylsilyl)oxy]methyl (TOM)(3,4,5) and 2'-O-tert.-butyldimethylsilyl (TBDMS) RNA synthesis methods. Recently, we have introduced synthetic routes to 2'-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, uridine, guanosine, cytidine, that are tailored for the 2'-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis method.(6).
Subject(s)
Crystallography, X-Ray , Oligoribonucleotides/chemical synthesis , Organoselenium Compounds/chemistry , Guanosine/analogs & derivatives , Guanosine/chemistry , Oligoribonucleotides/chemistryABSTRACT
Site-specifically modified 2'-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels-Alder ribozyme, have relied on this modification. Here, we introduce synthetic routes to 2'-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, cytidine, guanosine and uridine, that are tailored for 2'-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis. We additionally report on their incorporation into oligoribonucleotides including deprotection and purification. The methodological expansion of 2'-methylseleno labeling via ACE RNA chemistry is a major step to make Se-RNA generally accessible and to receive broad dissemination of the Se-approach for crystallographic studies on RNA. Thus far, preparation of 2'-methylseleno-modified oligoribonucleotides has been restricted to the 2'-O-[(triisopropylsilyl)oxy]methyl (TOM) and 2'-O-tert-butyldimethylsilyl (TBDMS) RNA synthesis methods.
Subject(s)
Crystallography, X-Ray , Oligoribonucleotides/chemical synthesis , Organophosphorus Compounds/chemistry , Organoselenium Compounds/chemical synthesis , Adenosine Monophosphate/analogs & derivatives , Biochemistry/methods , Cytidine Monophosphate/analogs & derivatives , Guanosine Monophosphate/analogs & derivatives , Oligoribonucleotides/chemistry , Organoselenium Compounds/chemistry , RNA/chemical synthesis , Uridine Monophosphate/analogs & derivativesABSTRACT
The derivatization of nucleic acids with selenium is a useful approach to facilitate phase determination during three-dimensional structural analysis by X-ray crystallography. This unit describes (1) the synthesis and characterization of 2'-deoxy-2'-methylseleno (2'-Se-methyl) nucleosides and their corresponding 3'-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite derivatives, (2) the site-specific incorporation of 2'-Se-methyl ribonucleosides into oligoribonucleotides by chemical RNA solid-phase synthesis, and (3) the enzymatic ligation of Se-containing RNA oligonucleotides to produce a biologically relevant RNA sequence.
Subject(s)
Amides/chemical synthesis , Organoselenium Compounds/chemistry , Phosphoric Acids/chemical synthesis , RNA/chemical synthesis , Amides/chemistry , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Nucleic Acid Conformation , Phosphoric Acids/chemistry , RNA/chemistryABSTRACT
We have recently introduced a basic concept for the combined chemical and enzymatic preparation of site-specifically modified 2'-methylseleno RNAs which represent useful derivatives for phasing of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established set of 2'-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis. The novel building block was readily incorporated into RNA. Importantly, it was the 2'-methylseleno-guanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization of selenium-containing RNA. In addition, we investigated 2'-methylseleno RNA with respect to crystallization properties. Our studies revealed that the Se modification significantly increases the range of conditions leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities for obtaining the best crystal form.
