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

Hikida, Yasushi; Kimoto, Michiko; Yokoyama, Shigeyuki; Hirao, Ichiro

doi:10.1038/nprot.2010.77

Cited by 45 publications

(29 citation statements)

References 43 publications

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“…Korlach et al [23] overcame similar problems in producing fluorescent-labeled deoxynucleotide triphosphate derivatives for use in sequencing DNA. Strategies under consideration include the use of mutant strains that would result in tRNAs containing a unique DHU position that can be labeled [16] as opposed to wild-type tRNAs, for which labeling is generally distributed over two or more DHU positions [24], [25]; and the introduction of fluorophores at other than DHU positions [22], [26], [27], [28]. Further improvements that would improve monitoring of more complex mixtures of mRNAs include decreasing the rates of dye photobleaching and of software algorithmic misidentification due to noise in the fluorescence intensity traces.…”

Section: Discussionmentioning

confidence: 99%

FRET-Based Identification of mRNAs Undergoing Translation

et al. 2012

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We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

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Section: Discussionmentioning

confidence: 99%

FRET-Based Identification of mRNAs Undergoing Translation

et al. 2012

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“…By introducing the s base at several positions within the tRNA, the magnesium ion dependency and the thermal stability of the L-shape structure formation were examined by the changes in the fluorescence of the s base at each position. 81,82) …”

Section: Hydrophobic Unnatural Base Pairsmentioning

confidence: 99%

Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma

Hirao

Kimoto

2012

Proc. Jpn. Acad., Ser. B

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Toward the expansion of the genetic alphabet of DNA, several artificial third base pairs (unnatural base pairs) have been created. Synthetic DNAs containing the unnatural base pairs can be amplified faithfully by PCR, along with the natural A–T and G–C pairs, and transcribed into RNA. The unnatural base pair systems now have high potential to open the door to next generation biotechnology. The creation of unnatural base pairs is a consequence of repeating “proof of concept” experiments. In the process, initially designed base pairs were modified to address their weak points. Some of them were artificially evolved to ones with higher efficiency and selectivity in polymerase reactions, while others were eliminated from the analysis. Here, we describe the process of unnatural base pair development, as well as the tests of their applications.

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“…Researchers are attempting to create expanded systems, and many unnatural base pairs have been designed and tested in in vitro biological systems [1][2][3][4][5]. Among them, some unnatural base pairs have exhibited high selectivity as a third base pair in PCR amplification and/or transcription [6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo [4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

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

Cited by 45 publications

References 43 publications

FRET-Based Identification of mRNAs Undergoing Translation

FRET-Based Identification of mRNAs Undergoing Translation

Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma

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

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