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Category: Microbial Genetics and Molecular Biology
Incorporation of Modified Nucleotides into RNA for Studies on RNA Structure, Function and Intermolecular Interactions, Page 1 of 2
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This chapter focuses on techniques for intentionally incorporating both natural and unnatural modified nucleotides into RNA molecules. The incorporation of modified nucleotides can also facilitate the structural analysis of RNAs and RNA-containing complexes. The chapter provides a broad overview of the types of experiments that can be performed with chemically modified RNAs. It describes the properties of particular modified nucleotides, and discusses techniques for their incorporation into polynucleotide chains. It presents a number of examples of the ways in which synthetic approaches have been used to investigate a variety of problems. It also illustrates the methods that pertain to individual areas of expertise. Many of the modified nucleotides retain the structural properties of their unmodified counterparts and can be randomly incorporated into polynucleotide chains by enzymatic synthesis. RNA molecules with modified nucleotides at predetermined sites can also be constructed by semisynthetic methods in which the modified residue is introduced at the 5' end of the 3' RNA fragment by transcription and then ligated to the 5' RNA fragment. A novel strategy for the site-specific incorporation of modified nucleotides makes use of circularly permuted RNAs (cpRNAs). The approaches described in this chapter will prove useful in the future in the many exciting areas of research involving naturally modified and edited RNAs.
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Modified nucleotides useful in RNA cross-linking experiments.
Modified nucleotides useful in RNA cross-linking experiments.
Structures of disulfide cross-links used in RNA structural studies.
Structures of disulfide cross-links used in RNA structural studies.
Base analogs useful in functional group analysis of RNA structures.
Base analogs useful in functional group analysis of RNA structures.
Sugar and phosphate analogs useful in analysis of RNA structures.
Sugar and phosphate analogs useful in analysis of RNA structures.
Steps involved in one cycle of solid-phase synthesis of oligoribonucleotides by the silyl-phosphoramidite method.
Steps involved in one cycle of solid-phase synthesis of oligoribonucleotides by the silyl-phosphoramidite method.
Transcription by T7 RNA polymerase of plasmid DNA (a), amplified DNA (b), and synthetic DNA (c) templates. Black rectangles denote the 17-bp T7 RNA polymerase promoter; RS signifies a restriction site.
Transcription by T7 RNA polymerase of plasmid DNA (a), amplified DNA (b), and synthetic DNA (c) templates. Black rectangles denote the 17-bp T7 RNA polymerase promoter; RS signifies a restriction site.
Construction of recombinant RNA molecules containing modified nucleotides, (a) Replacement of A73 and A76 of yeast tRNAPhe with 2-azidoadenosine (2N3A). Whitfeld degradation entails repeated cycles of periodate oxidation, aniline cleavage and phosphatase treatment. ( Adapted from Wower et al., 1988. ) (b) Substitution for Y37 of yeast tRNAPhe by 2- or 8-azidoadenosine (N3A). ( Adapted from Bruce and Uhlenbeck, 1982 , and Sylvers et al., 1992 .) (c) Introduction of azidoadenosine (N,A) at position 21 of yeast tRNAPhe by using a chimeric oligonucleotide to direct cleavage by RNase Η and the DNA ligase method to reconstruct the intact tRNA molecule. ( Adapted from Wower et al., 1994a. ) (d) Semisynthetic incorporation of 4-thioU (s4U) into a long RNA molecule. Black rectangles indicate the promoter for T7 RNA polymerase. Transcripts are joined by the DNA ligase method. (Adapted from Sontheimer, 1994 .) (e) Site-specific incorporation of modified nucleotides through the use of circularly permuted RNAs (cpRNAs). Black rectangle, T7 RNA polymerase promoter; GMPS, guanosine 5′-monophosphorothioate; APAB, p-azidophenacyl bromide; ΑΡΑ, p-azidophenacyl moiety. (Adapted from Nolan et al., 1993 .) Asterisks indicate positions labeled with 32P.
Construction of recombinant RNA molecules containing modified nucleotides, (a) Replacement of A73 and A76 of yeast tRNAPhe with 2-azidoadenosine (2N3A). Whitfeld degradation entails repeated cycles of periodate oxidation, aniline cleavage and phosphatase treatment. ( Adapted from Wower et al., 1988. ) (b) Substitution for Y37 of yeast tRNAPhe by 2- or 8-azidoadenosine (N3A). ( Adapted from Bruce and Uhlenbeck, 1982 , and Sylvers et al., 1992 .) (c) Introduction of azidoadenosine (N,A) at position 21 of yeast tRNAPhe by using a chimeric oligonucleotide to direct cleavage by RNase Η and the DNA ligase method to reconstruct the intact tRNA molecule. ( Adapted from Wower et al., 1994a. ) (d) Semisynthetic incorporation of 4-thioU (s4U) into a long RNA molecule. Black rectangles indicate the promoter for T7 RNA polymerase. Transcripts are joined by the DNA ligase method. (Adapted from Sontheimer, 1994 .) (e) Site-specific incorporation of modified nucleotides through the use of circularly permuted RNAs (cpRNAs). Black rectangle, T7 RNA polymerase promoter; GMPS, guanosine 5′-monophosphorothioate; APAB, p-azidophenacyl bromide; ΑΡΑ, p-azidophenacyl moiety. (Adapted from Nolan et al., 1993 .) Asterisks indicate positions labeled with 32P.
Mechanisms of RNA joining reactions catalyzed by T4 RNA ligase (A) and T4 DNA ligase (B).
Mechanisms of RNA joining reactions catalyzed by T4 RNA ligase (A) and T4 DNA ligase (B).
Template-dependent chemical ligation to form an oligoribonucleotide containing a trisubstituted pyrophosphate linkage.
Template-dependent chemical ligation to form an oligoribonucleotide containing a trisubstituted pyrophosphate linkage.
The apex region of the TAR RNA stem-loop, showing sites where chemical substitution or interference has been used to determine functionalities important in recognition by HIV-1 Tat.
The apex region of the TAR RNA stem-loop, showing sites where chemical substitution or interference has been used to determine functionalities important in recognition by HIV-1 Tat.
Summary of cross-links formed between mRNA analogs and three regions of the 16S rRNA in 30S ribosomal subunits. (Data from Rinke-Appel et al., 1991 , 1993 , 1994 ; Dontsova et al., 1992a ; and Sergiev et al, 1997 .)
Summary of cross-links formed between mRNA analogs and three regions of the 16S rRNA in 30S ribosomal subunits. (Data from Rinke-Appel et al., 1991 , 1993 , 1994 ; Dontsova et al., 1992a ; and Sergiev et al, 1997 .)
Short-range cross-links formed between tRNA and the A, Ρ and Ε sites of the E. coli ribosome. SX, 30S subunit proteins; LX, 50S subunit proteins; arabic numerals, nucleotides in 23S rRNA; italic numerals, nucleotides in 16S rRNA. (Data from Prince et al., 1982 ; Wower et al., 1989 , 1990 , 1993a , and 1995 ; Sylvers et al., 1992 ; Rosen et al,, 1993 ; and Rosen and Zimmermann, 1997 and unpublished data.)
Short-range cross-links formed between tRNA and the A, Ρ and Ε sites of the E. coli ribosome. SX, 30S subunit proteins; LX, 50S subunit proteins; arabic numerals, nucleotides in 23S rRNA; italic numerals, nucleotides in 16S rRNA. (Data from Prince et al., 1982 ; Wower et al., 1989 , 1990 , 1993a , and 1995 ; Sylvers et al., 1992 ; Rosen et al,, 1993 ; and Rosen and Zimmermann, 1997 and unpublished data.)