PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification andDsand pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modifiedPabases into RNA by T7 transcription. To prepareDs-containing DNA templates with long chains, theDs-Pxpair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containingDsat specific positions. Using theseDs-containing DNA templates and a biotin-linkedPasubstrate (Biotin-PaTP) as a modifiedPabase, 260-mer RNA transcripts containing Biotin-Paat a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining theDs-PxandDs-Papairs with modifiedPasubstrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

Download Full-text

Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

Molecules ◽

10.3390/molecules17032855 ◽

2012 ◽

Vol 17 (3) ◽

pp. 2855-2876 ◽

Cited By ~ 30

Author(s):

Nobuyuki Morohashi ◽

Michiko Kimoto ◽

Akira Sato ◽

Rie Kawai ◽

Ichiro Hirao

Keyword(s):

Base Pair ◽

Site Specific ◽

Transcription System ◽

Functional Components

Download Full-text

Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry

Chemical Communications ◽

10.1039/c2cc36293g ◽

2012 ◽

Vol 48 (88) ◽

pp. 10835 ◽

Cited By ~ 42

Author(s):

Takumi Ishizuka ◽

Michiko Kimoto ◽

Akira Sato ◽

Ichiro Hirao

Keyword(s):

Click Chemistry ◽

Base Pair ◽

Site Specific ◽

Rna Molecules ◽

Transcription System

Download Full-text

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs

Nucleic Acids Research ◽

10.1093/nar/gni128 ◽

2005 ◽

Vol 33 (15) ◽

pp. e129-e129 ◽

Cited By ~ 49

Author(s):

K. Moriyama ◽

M. Kimoto ◽

T. Mitsui ◽

S. Yokoyama ◽

I. Hirao

Keyword(s):

Base Pairs ◽

Site Specific ◽

Rna Molecules

Download Full-text

Interaction of influenza virus polymerase with viral RNA in the ‘corkscrew’ conformation

Journal of General Virology ◽

10.1099/0022-1317-80-10-2565 ◽

1999 ◽

Vol 80 (10) ◽

pp. 2565-2572 ◽

Cited By ~ 71

Author(s):

Ramon Flick ◽

Gerd Hobom

Keyword(s):

Influenza Virus ◽

Base Pair ◽

Short Range ◽

Nucleotide Substitutions ◽

Base Pairs ◽

Stem Loop ◽

Rna Molecules ◽

Virus Rna ◽

Core Elements ◽

Complete Set

The influenza virus RNA (vRNA) promoter structure is known to consist of the 5′- and 3′-terminal sequences of the RNA, within very narrow boundaries of 16 and 15 nucleotides, respectively. A complete set of single nucleotide substitutions led to the previously proposed model of a binary hooked or ‘corkscrew’ conformation for the vRNA promoter when it interacts with the viral polymerase. This functional structure is confirmed here with a complete set of complementary double substitutions, of both the regular A:U and G:C type and also the G:U type of base-pair exchanges. The proposed structure consists of a six base-pair RNA rod in the distal element in conjunction with two stem–loop structures of two short-range base- pairs (positions 2–9; 3–8). These support an exposed tetranucleotide loop within each branch of the proximal element, in an overall oblique organization due to a central unpaired A residue at position 10 in the 5′ sequence. Long-range base-pairing between the entire 5′ and 3′ branches, as required for an unmodified ‘panhandle’ model, has been excluded for the proximal element, while it is known to represent the mode of interaction within the distal element. A large number of short-range base-pair exchanges in the proximal element constitute promoter-up mutations, which show activities several times above that of the wild- type in reporter gene assays. The unique overall conformation and rather few invariant nucleotides appear to be the core elements in vRNA recognition by polymerase and also in viral ribonucleoprotein packaging, to allow discrimination against the background of other RNA molecules in the cell.

Download Full-text

Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

Australian Journal of Chemistry ◽

10.1071/ch05196 ◽

2005 ◽

Vol 58 (12) ◽

pp. 851 ◽

Cited By ~ 1

Author(s):

Philip Hendry ◽

Maxine J. McCall ◽

Trevor J. Lockett

Keyword(s):

Optimal Design ◽

Base Pair ◽

Hammerhead Ribozymes ◽

Base Pairs ◽

Physiological Conditions ◽

Rna Transcripts ◽

Cleavage Efficiency ◽

Double Helices

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.

Download Full-text

Faculty Opinions recommendation of Site-specific incorporation of functional components into RNA by transcription using unnatural base pair systems.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1471957.951055 ◽

2010 ◽

Author(s):

Michael Yarus

Keyword(s):

Base Pair ◽

Site Specific ◽

Functional Components

Download Full-text

Unnatural base pairs mediate the site-specific incorporation of an unnatural hydrophobic component into RNA transcripts

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2004.02.072 ◽

2004 ◽

Vol 14 (10) ◽

pp. 2593-2596 ◽

Cited By ~ 13

Author(s):

Masayuki Endo ◽

Tsuneo Mitsui ◽

Taeko Okuni ◽

Michiko Kimoto ◽

Ichiro Hirao ◽

...

Keyword(s):

Base Pairs ◽

Site Specific ◽

Rna Transcripts ◽

Hydrophobic Component

Download Full-text

Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4721-4727.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4721-4727 ◽

Cited By ~ 27

Author(s):

Stefan J. Green ◽

Dror Minz

Keyword(s):

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

Pcr Amplification ◽

Environmental Dna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Dna Templates ◽

Target Dna ◽

Gradient Gel Electrophoresis ◽

Pcr Method

ABSTRACT PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.

Download Full-text