Method for synthesizing selectively labeled RNA
Inventors
Wang, Yun-Xing • Liu, Yu • Sousa, Rui
Assignees
University of Texas System • US Department of Health and Human Services • Government of the United States of America
Publication Number
US-11041182-B2
Publication Date
2021-06-22
Expiration Date
2034-07-08
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Abstract
The invention relates to a method for synthesizing a selectively labeled RNA, and an apparatus for performing the method. Specific segments or discrete residues within the RNA may be selectively labeled, and different segments may include different labels.
Core Innovation
The invention relates to a method for synthesizing a selectively labeled RNA and an apparatus for performing the method. The method allows specific segments or discrete residues within the RNA to be selectively labeled, and different segments may include different labels. This is achieved by a hybrid solid-liquid phase enzymatic method that integrates initiation, elongation, and termination stages where RNA synthesis is paused by controlling temperature and the composition of ribonucleoside triphosphates (rNTPs) to stabilize a ternary complex comprising the RNA polymerase, DNA template attached to a solid substrate, and RNA being synthesized.
The problem being solved arises from limitations of existing RNA synthesis methods. Solution-based T7 transcription can generate long RNA molecules in large quantity but cannot selectively label specific residues or segments. Chemical synthesis is limited to short RNAs in minute quantities and is impractical for selectively labeling sizable RNAs due to low coupling efficiency and high cost. The inventive method overcomes these problems by enabling selective labeling at designated residues or segments with high efficiency similar to that of solution-phase T7 transcription, using stable isotope labels or fluorophores.
The method involves providing a solid phase comprising a DNA template attached to a solid substrate, and mixing it with a liquid phase containing RNA polymerase and specific rNTPs. RNA synthesis is initiated, then paused by lowering the temperature and manipulating rNTP content, allowing separation of solid and liquid phases. Different liquid phases with various labeled rNTP mixtures are sequentially applied to elongate the RNA in steps, enabling selective segment labeling. The method includes gentle mixing to prevent formation of bubbles, and use of rNTP concentrations approximately stoichiometric relative to the DNA template, which is counter to conventional approaches.
Claims Coverage
The patent claims three independent claims covering a method for synthesizing RNA through repeated initiation, elongation, and termination stages, with selective labeling of RNA.
Method for synthesis of RNA using hybrid solid-liquid phases
A multi-stage RNA synthesis method comprising initiation, elongation, and termination stages, wherein a DNA template attached to a solid substrate is mixed with liquid phases containing RNA polymerase and rNTPs, incubated at 4-37° C. for synthesis, then RNA synthesis is paused by incubating at 0-5° C. for at least 10 minutes to form a ternary complex, and phases are separated allowing sequential steps.
Controlled elongation with cycles incorporating different rNTP mixtures
The elongation stage involves repeated cycles of mixing the solid phase with liquid phases containing rNTPs (labeled or unlabeled), incubation at 4-37° C. to elongate RNA, pausing at low temperature 0-5° C., separating solid and liquid phases, with the rNTPs in each cycle potentially differing to enable selective segment labeling, repeated up to 100 times.
Termination stage to prevent re-initiation and termination of synthesis
The termination stage involves mixing the solid phase with a third liquid phase containing rNTPs (potentially missing the first nucleotide required to initiate new synthesis), incubation at 4-37° C., followed by pausing at 0° C., repeated multiple times to control the yield of selectively labeled RNA, with labeling possible in any of the liquid phases.
The claims cover a method for synthesizing RNA by cyclically performing initiation, elongation, and termination stages using a solid phase DNA template with RNA polymerase and rNTPs, with controlled pauses to separate phases, allowing incorporation of labels selectively in segments by varying rNTPs and incubation conditions.
Stated Advantages
Allows specific labeling of residues and segments within RNA similarly efficient as solution-phase T7 transcription.
Enables synthesis of sizable selectively labeled RNA segments impractical with chemical synthesis due to low yield and high cost.
Provides flexibility in labeling different RNA segments with different isotopes, fluorophores, or chemical modifications.
Documented Applications
Applications in structural studies using NMR spectroscopy.
Determining phase in X-ray crystallography.
RNA-aptamer-based detection of substances, bacteria, or viral particles.
Disease diagnosis using RNA-based reagents.
Use in single molecule FRET (sFRET) analysis.
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