Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis

2010 ◽  
Vol 5 (7) ◽  
pp. 1312-1323 ◽  
Author(s):  
Yasushi Hikida ◽  
Michiko Kimoto ◽  
Shigeyuki Yokoyama ◽  
Ichiro Hirao
Keyword(s):  
Base Pair ◽  
Conformation Analysis ◽  
Site Specific ◽  
Rna Molecules ◽  
Structural Conformation

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

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Michiko Kimoto ◽  
Rie Yamashige ◽  
Shigeyuki Yokoyama ◽  
Ichiro Hirao
Keyword(s):  
Base Pair ◽  
Pcr Amplification ◽  
Base Pairs ◽  
Dna Templates ◽  
Site Specific ◽  
Rna Molecules ◽  
Transcription System ◽  
Rna Transcripts ◽  
Pcr Method ◽  
Functional Components

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.

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

2012 ◽  
Vol 48 (88) ◽  
pp. 10835 ◽  
Author(s):  
Takumi Ishizuka ◽  
Michiko Kimoto ◽  
Akira Sato ◽  
Ichiro Hirao
Keyword(s):  
Click Chemistry ◽  
Base Pair ◽  
Site Specific ◽  
Rna Molecules ◽  
Transcription System

scholarly journals Supercoiling and looping promote DNA base accessibility and coordination among distant sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan M. Fogg ◽  
Allison K. Judge ◽  
Erik Stricker ◽  
Hilda L. Chan ◽  
Lynn Zechiedrich
Keyword(s):  
Mechanical Stress ◽  
Genomic Instability ◽  
Base Pair ◽  
Base Pairing ◽  
Complex Interplay ◽  
Site Specific ◽  
Torsional Strain ◽  
Dna Base ◽  
Dna Backbone ◽  
Negative Supercoiling

AbstractDNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity.

scholarly journals Site-Specific Chemical Labeling of Long RNA Molecules

2011 ◽  
Vol 22 (1) ◽  
pp. 95-100 ◽  
Author(s):  
Kasper Jahn ◽  
Eva Maria Olsen ◽  
Morten Muhlig Nielsen ◽  
Thomas Tørring ◽  
Reza MohammadZadegan ◽  
...  
Keyword(s):  
Chemical Labeling ◽  
Specific Chemical ◽  
Site Specific ◽  
Rna Molecules

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

2005 ◽  
Vol 33 (15) ◽  
pp. e129-e129 ◽  
Author(s):  
K. Moriyama ◽  
M. Kimoto ◽  
T. Mitsui ◽  
S. Yokoyama ◽  
I. Hirao
Keyword(s):  
Base Pairs ◽  
Site Specific ◽  
Rna Molecules

scholarly journals Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

1986 ◽  
Vol 6 (12) ◽  
pp. 4329-4334 ◽  
Author(s):  
P V Prasad ◽  
D Horensky ◽  
L J Young ◽  
M Jayaram
Keyword(s):  
Base Pair ◽  
Substrate Recognition ◽  
Binding Assays ◽  
Site Specific ◽  
Contact Points ◽  
Dyad Symmetry

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.

scholarly journals Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

2020 ◽  
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Base Pair ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

AbstractConjugation of RNAs with nanoparticles is of significant importance for its numerous applications in biology and medicine, which however remains challenging, especially for large ones. So far, the majority of RNA labeling rely on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nts. We here present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with gold nanoparticle (AuNP) empowered by expanded genetic alphabet transcription. We synthesize an amine-derivatized TPT3 (TPT3A), which are site-specifically incorporated into a 97-nt 3’SL RNA and a 719-nt mini genomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by standard in vitro transcription with expanded genetic alphabet containing the A-T, G-C natural base pairs and the TPT3-NaM unnatural base pair. TPT3 modification cause minimal structural perturbations to the RNAs by small angle X-ray scattering. The purified TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-NHS-Nanogold nanoparticles via the highly selective amine-NHS ester reaction and purified under non-denaturing conditions. We demonstrate the application of the AuNP-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The inter-nanoparticle distance distributions in the 3’SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus validate the applicability of this novel labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies, and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.Significance StatementWe present a site-specific labeling strategy for large RNAs by T7 transcription with expanded genetic alphabet containing TPT3-NaM unnatural base pair. The applicability of this labeling strategy is validated by X-ray scattering interferometry measurements on a 97-nt and a 719-nt RNAs. This strategy can be applicable to natural RNAs or artificial RNA nanostructures with sizes from tens up to thousands of nucleotides, or covalent conjugation of RNAs with other metal nanoparticles. The usage of a far upstream forward primer during PCR enables easy purification of RNA from DNA templates, the non-denaturing conditions for conjugation reactions and purification avoids potential large RNA misfolding. This labeling strategy expands our capability to site-specifically conjugate RNA with nanoparticles for many applications.

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

1999 ◽  
Vol 80 (10) ◽  
pp. 2565-2572 ◽  
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.

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