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 • Office of Technology Transfer
Publication Number
US-10190143-B2
Publication Date
2019-01-29
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 provides a method and apparatus for synthesizing selectively labeled RNA, allowing specific segments or discrete residues within the RNA to be selectively labeled with different labels. This hybrid solid-liquid phase enzymatic method enables specific labeling at designated residue(s) and/or segment(s) of an RNA, including labeling with stable isotopes such as 13C/15N or fluorophores like Cy3 and Cy5 rCTP, rUTP, or rNTP derivatives.
The problem addressed is that conventional RNA synthesis methods either produce large quantities of RNA without selective labeling (solution-based T7 transcription) or small quantities of selectively labeled RNA with low efficiency and high cost (chemical synthesis). Specifically labeling sizable RNAs using chemical synthesis is impractical due to extremely low efficiency and prohibitive cost. The invention overcomes these limitations by providing a method with labeling efficiency similar to solution-phase T7 transcription, but with the ability to selectively label specific residues or segments.
The method involves performing an initiation stage, an elongation stage, and a termination stage. The initiation stage includes providing a solid phase with DNA templates attached to solid substrates, mixing with a liquid phase containing RNA polymerase and rNTPs, incubating to initiate RNA synthesis, pausing RNA synthesis by lowering the temperature, and separating the solid phase from the liquid phase. The elongation stage comprises cycles of mixing the solid phase with liquid phases containing different rNTPs (which may be differently labeled), incubating, pausing, separating, and repeating to elongate the RNA with selective labeling. The termination stage similarly mixes the solid phase with rNTPs to conclude synthesis. The rNTPs in any of the liquid phases may include labels, enabling selective segmental labeling.
Claims Coverage
The patent includes one main independent claim defining a method for synthesizing RNA with selective labeling, supported by additional claims specifying substrate type, reaction conditions, enzymes, labeling, and apparatus details. There is one independent claim overall.
Hybrid solid-liquid phase RNA synthesis method with staged initiation, elongation, and termination
The method comprises performing an initiation stage with a solid phase DNA template attached to a solid substrate combined with a liquid phase containing RNA polymerase and rNTPs, incubating to initiate RNA synthesis, pausing synthesis at reduced temperatures, separating phases; an elongation stage repeating cycles of incubating with different rNTP mixtures at 4-37°C, pausing at 0-5°C, separating phases, repeated 1-100 times with potentially different rNTPs in each cycle; and a termination stage incubating with rNTPs and pausing synthesis at 0°C.
Use of solid substrate beads for DNA templates
The DNA templates are attached to solid substrates that are beads comprising gel, glass, or synthetic polymers, with bead diameters of 5-100 μm.
Controlled rNTP to DNA concentration ratio
The concentration ratio of rNTPs to DNA is maintained in the range of 1-100 to optimize reaction conditions.
Use of T7 RNA polymerase
The RNA polymerase employed in the method is T7 RNA polymerase.
Incorporation of diverse labels into rNTPs
Labels in rNTPs include 13C/15N, 2H, Cy3, Cy5, fluorophores, heavy atoms, or chemical modifications, allowing selective incorporation into synthesized RNA.
Temperature and mixing controls to pause and process the synthesis
Pausing RNA synthesis is controlled by incubating at low temperatures (0-5°C) and by omitting specific rNTPs to control elongation. Mixing is performed so as not to create bubbles, including rotating the reaction 360 degrees under inert atmosphere conditions.
Automated apparatus for performing RNA synthesis
An automated platform is provided comprising reaction vessels manipulated by motors and holders to gently mix reactions without stirring, shaking, or bubbling. This apparatus is computer controlled and integrates components for gentle sample handling and phase separation during RNA synthesis.
The claims define a method with staged RNA synthesis using a hybrid solid-liquid phase approach enabling selective RNA labeling, enabled by control of reaction conditions, substrates, enzymes, labeling moieties, and an apparatus for gentle automated processing.
Stated Advantages
Allows specific labeling of RNA segments or discrete residues efficiently, similar to solution-phase transcription, overcoming inefficiencies and cost of chemical synthesis for large RNA.
Enables selective incorporation of diverse labels including stable isotopes and fluorophores for use in structural studies and detection.
Method permits pausing and restarting RNA synthesis at defined positions, enabling segmented labeling.
Automated platform supports gentle mixing and control, increasing reproducibility and preventing reaction damage such as bubble formation.
Documented Applications
Structural studies using nuclear magnetic resonance (NMR).
Determining phase in X-ray crystallography.
RNA-aptamer-based detection of substances, bacteria, or viral particles.
Disease diagnosis based on selectively labeled RNA reagents.
Single molecule FRET analysis using specifically labeled RNA.
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