mRNA Assembly and Modifications via synthetic RNA oligonucleotides, Advanced Cell and Immune Therapy
Luke McLaughlin, Biotech Digital Marketer, Business Developer and Life Science Content Creator
mRNA (messenger RNA) plays a crucial role in translating genetic information from DNA into proteins. In the field of biotechnology, enhancing the stability, functionality, and detection of mRNA in vivo is paramount for various applications, including gene therapy, vaccine development, and basic research. This article delves into the techniques for modifying mRNA nucleobases and backbones to achieve these goals and outlines methods for assembling kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides.
In the realm of molecular biology, the quest to stabilize and optimize mRNA for therapeutic and research purposes has led to innovative modifications at both the nucleobase and backbone levels. These modifications enhance mRNA's resistance to degradation, improve its translational efficiency, and allow for precise tracking and manipulation within biological systems. This article will explore these modifications in detail, followed by a comprehensive look at the methods used to assemble Kb-length mRNA from shorter RNA sequences.
Methods of Assembling Kb-Length mRNA from Shorter RNA Oligonucleotides
T4 RNA Ligase-Based Assembly
T4 RNA ligase-based assembly is a widely used technique for assembling kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides. This method leverages the ability of T4 RNA ligase to catalyze the formation of phosphodiester bonds between RNA molecules, enabling the creation of long, continuous RNA sequences from shorter fragments. Here is a detailed breakdown of the process:
Design and Synthesis
Overlapping RNA Oligonucleotides:
Length and Overlap: Typically, RNA oligonucleotides of about 70 nucleotides (70mers) are synthesized. These oligonucleotides are designed to have 20-30 nucleotide overlaps with adjacent oligonucleotides. The overlap ensures proper annealing and alignment for subsequent ligation.
Sequence Design: Careful consideration is given to the sequence design to avoid secondary structures that could interfere with annealing and ligation. This often involves using sequence analysis software to predict and minimize the formation of hairpins or other stable structures.
Chemical Synthesis:
Phosphoramidite Chemistry: RNA oligonucleotides are synthesized using automated synthesizers based on phosphoramidite chemistry. This method allows for the sequential addition of nucleotides to a growing chain, providing precise control over the sequence.
Quality Control: After synthesis, the oligonucleotides are purified, typically using high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE), to ensure high purity and correct length.
Annealing
Equimolar Mixing:
Equimolar Ratios: The RNA oligonucleotides are mixed in equimolar ratios to ensure that each fragment has an equal chance of finding its complementary overlap partner. This balance is crucial for efficient and accurate assembly.
Gradual Cooling:
Denaturation and Annealing: The mixture is first heated to a high temperature (typically around 95°C) to denature any secondary structures. The temperature is then gradually lowered to promote annealing. A typical annealing protocol might involve cooling from 95°C to room temperature over several hours.
Buffer Conditions: The annealing buffer usually contains salts (e.g., sodium or potassium ions) to stabilize the hybridized RNA duplexes, and sometimes magnesium ions (Mg^2+) to further stabilize RNA-RNA interactions.
Ligation
T4 RNA Ligase:
Enzyme Source: T4 RNA ligase, derived from bacteriophage T4, is the enzyme of choice due to its high efficiency in ligating RNA ends.
Catalysis: The enzyme catalyzes the formation of phosphodiester bonds between the 3’-hydroxyl group of one RNA fragment and the 5’-phosphate group of another. ATP is required as a cofactor for the ligation reaction.
Reaction Conditions:
Optimal Temperature: The ligation reaction is typically performed at a low temperature (around 16°C) to stabilize the RNA duplexes and minimize the formation of secondary structures that could hinder ligation.
Buffer Composition: The ligation buffer generally includes Tris-HCl (to maintain pH), MgCl_2 (as a divalent cation necessary for enzymatic activity), ATP (as an energy source), and DTT (dithiothreitol, to maintain reducing conditions).
Ligation Efficiency:
Time: The ligation reaction is usually allowed to proceed overnight to maximize the yield of full-length RNA.
Concentration: High concentrations of RNA oligonucleotides (typically in the micromolar range) are used to drive the ligation reaction forward.
Purification
Gel Electrophoresis:
Denaturing PAGE: The ligated RNA is purified using denaturing polyacrylamide gel electrophoresis (PAGE). Denaturing conditions (using urea) prevent secondary structures and ensure separation based on size.
Visualization: RNA bands are visualized using UV shadowing or staining with dyes such as ethidium bromide or SYBR Green.
Extraction: The desired full-length RNA band is excised from the gel, and the RNA is extracted by crushing the gel slice, soaking in an elution buffer, and then precipitating the RNA with ethanol.
Column Chromatography:
Affinity Chromatography: Alternatively, column chromatography methods, such as anion-exchange or affinity chromatography, can be used to purify the RNA. Columns with resins that selectively bind RNA can help isolate the full-length product from shorter fragments and other impurities.
Elution: The RNA is eluted from the column using a gradient of increasing ionic strength or specific elution buffers designed to release the bound RNA.
Example Protocol for T4 RNA Ligase-Based Assembly
Materials and Reagents:
Synthetic RNA oligonucleotides (70mers) with designed overlaps
T4 RNA ligase
ATP (100 mM stock solution)
Ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl_2, 10 mM DTT)
Denaturing PAGE apparatus and reagents
Gel extraction kit or appropriate elution buffers
Ethanol and salts for RNA precipitation
Procedure:
Mix RNA Oligonucleotides:
Combine the RNA oligonucleotides in equimolar amounts in a microcentrifuge tube.
Add annealing buffer to a final concentration of 1x (e.g., 10 mM Tris-HCl, pH 7.5, 50 mM NaCl).
Denature and Anneal:
Heat the mixture to 95°C for 2 minutes to denature secondary structures.
Gradually cool the mixture to room temperature over 2-3 hours to allow annealing.
Prepare Ligation Reaction:
In a new tube, set up the ligation reaction by combining the annealed RNA mixture, T4 RNA ligase, ATP, and ligation buffer.
Incubate the reaction mixture at 16°C overnight.
Purify Ligated RNA:
Run the ligation mixture on a denaturing polyacrylamide gel (typically 8-10% polyacrylamide with 7 M urea).
Visualize the RNA bands and excise the full-length mRNA band.
Extract the RNA from the gel slice using a gel extraction kit or by eluting in an appropriate buffer.
Final Purification:
Precipitate the RNA by adding 2.5 volumes of ethanol and 0.1 volumes of 3 M sodium acetate, pH 5.2.
Incubate at -20°C for at least 30 minutes, then centrifuge to pellet the RNA.
Wash the pellet with 70% ethanol, air dry, and resuspend in RNase-free water.
T4 RNA ligase-based assembly is a powerful method for creating long mRNA sequences from shorter RNA oligonucleotides. By carefully designing overlapping sequences, optimizing annealing and ligation conditions, and employing effective purification techniques, researchers can produce high-quality, full-length mRNA suitable for various applications in molecular biology and biotechnology. The ability to customize and modify these RNA sequences opens up a wide array of possibilities for studying gene expression, developing therapeutic interventions, and advancing our understanding of RNA biology.
Template-Directed Synthesis Using T7 RNA Polymerase
Template-Directed Synthesis Using T7 RNA Polymerase: Technical Details
Template-directed synthesis using T7 RNA polymerase is a robust method for generating kilobase-length (Kb-length) mRNA from overlapping RNA oligonucleotides. This method involves creating a single-stranded DNA (ssDNA) template from which T7 RNA polymerase can transcribe the desired mRNA. Below is a detailed breakdown of each step involved in this process.
Template Preparation
Assembling Overlapping RNA Oligonucleotides into a DNA Template:
Design and Synthesis of Oligonucleotides: The process begins with designing and synthesizing overlapping RNA oligonucleotides (typically 70mers) with regions of 20-30 nucleotide overlaps. These overlapping sequences ensure that when the oligonucleotides anneal, they form a continuous template.
Overlap Extension PCR (OE-PCR): The overlapping RNA oligonucleotides are converted into a single-stranded DNA (ssDNA) template using overlap extension PCR. This involves two main steps: the assembly of oligonucleotides into a longer DNA sequence and the amplification of this assembled sequence.
Procedure for Overlap Extension PCR:
Annealing and Extension:
Mix Oligonucleotides: Combine equimolar amounts of overlapping RNA oligonucleotides in a PCR tube.
Initial Annealing: Heat the mixture to 95°C for 2 minutes to denature any secondary structures, then slowly cool to 50-60°C (depending on the melting temperature of the oligonucleotides) to allow the overlapping regions to anneal.
Extension: Add DNA polymerase (e.g., Taq polymerase) and deoxynucleotide triphosphates (dNTPs) to the annealed oligonucleotide mixture. The polymerase extends the overlapping oligonucleotides, creating a continuous DNA strand.
Amplification:
PCR Reaction: Amplify the assembled DNA template using PCR. This involves repeated cycles of denaturation (95°C), annealing (50-60°C), and extension (72°C).
Primers: Use primers complementary to the 5’ and 3’ ends of the assembled template to initiate amplification. These primers ensure the specific and efficient amplification of the desired DNA template.
Purification:
Gel Electrophoresis: Purify the amplified DNA template using agarose gel electrophoresis to separate the desired product from any incomplete or nonspecific products.
Extraction: Excise the band corresponding to the full-length DNA template and extract the DNA using a gel extraction kit.
In Vitro Transcription
Using T7 RNA Polymerase to Synthesize mRNA:
Preparation of the Transcription Reaction: The purified ssDNA template is used as the template for in vitro transcription by T7 RNA polymerase. This enzyme recognizes the T7 promoter sequence, which is typically included in the design of the DNA template, and transcribes RNA from this template.
Procedure for In Vitro Transcription:
Set Up the Transcription Reaction:
Reaction Mix: Combine the following components in a reaction tube:
Purified ssDNA template
T7 RNA polymerase
NTPs (ATP, CTP, GTP, UTP)
Transcription buffer (typically containing Tris-HCl, MgCl_2, DTT, and spermidine)
Template Concentration: The concentration of the DNA template is typically in the range of 0.1-1 µg/µL to ensure efficient transcription.
Incubation:
Temperature: Incubate the reaction at 37°C for 2-4 hours. This temperature is optimal for T7 RNA polymerase activity.
Reaction Volume: The total reaction volume can vary but is usually around 20-100 µL, depending on the desired yield.
Optional DNase Treatment:
Removal of Template DNA: After transcription, treat the reaction mixture with DNase I to degrade the DNA template. This step ensures that the final product is free of contaminating DNA.
Purification
Purification of Synthesized mRNA:
Phenol-Chloroform Extraction:
Phase Separation: Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) to the transcription reaction and vortex to mix. Centrifuge the mixture to separate the aqueous (RNA-containing) phase from the organic phase.
Aqueous Phase Recovery: Carefully transfer the aqueous phase to a new tube.
Ethanol Precipitation:
Precipitation: Add 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol to the aqueous phase. Mix and incubate at -20°C for at least 30 minutes to precipitate the RNA.
Pelleting: Centrifuge the mixture at high speed (e.g., 12,000 x g) for 15 minutes at 4°C to pellet the RNA.
Washing: Wash the RNA pellet with 70% ethanol to remove any residual salts and contaminants. Centrifuge again to pellet the RNA.
Resuspension: Air dry the RNA pellet and resuspend it in RNase-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).
Example Protocol for Template-Directed Synthesis Using T7 RNA Polymerase
Materials and Reagents:
Synthetic RNA oligonucleotides with overlapping sequences
DNA polymerase (e.g., Taq polymerase)
dNTPs (100 mM stock solutions of each nucleotide)
Primers complementary to the ends of the assembled template
T7 RNA polymerase
NTPs (100 mM stock solutions of ATP, CTP, GTP, and UTP)
Transcription buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl_2, 2 mM spermidine, 10 mM DTT)
DNase I
Phenol-chloroform-isoamyl alcohol (25:24:1)
Ethanol and sodium acetate for RNA precipitation
RNase-free water or TE buffer
Procedure:
Assemble DNA Template:
Mix equimolar amounts of overlapping RNA oligonucleotides in a PCR tube.
Heat to 95°C for 2 minutes, then gradually cool to 50-60°C to anneal the oligonucleotides.
Add DNA polymerase, dNTPs, and extension buffer. Perform extension at 72°C for 5-10 minutes.
Amplify DNA Template:
Set up a PCR reaction with the annealed product, primers, DNA polymerase, dNTPs, and PCR buffer.
Perform 25-35 cycles of PCR: denaturation at 95°C for 30 seconds, annealing at 50-60°C for 30 seconds, and extension at 72°C for 1 minute.
Purify the amplified product using agarose gel electrophoresis and a gel extraction kit.
Transcribe RNA:
Combine the purified DNA template with T7 RNA polymerase, NTPs, and transcription buffer in a reaction tube.
Incubate at 37°C for 2-4 hours.
Optionally, treat with DNase I to remove the DNA template.
Purify RNA:
Extract the RNA using phenol-chloroform-isoamyl alcohol. Transfer the aqueous phase to a new tube.
Precipitate the RNA with sodium acetate and ethanol. Incubate at -20°C for 30 minutes.
Centrifuge to pellet the RNA, wash with 70% ethanol, and resuspend in RNase-free water or TE buffer.
Template-directed synthesis using T7 RNA polymerase is a powerful technique for producing long RNA sequences from shorter oligonucleotides. By leveraging the high fidelity and efficiency of T7 RNA polymerase, researchers can generate high-quality mRNA for various applications, including gene expression studies, vaccine development, and therapeutic interventions. The detailed steps outlined above highlight the critical aspects of template preparation, in vitro transcription, and purification, ensuring the successful synthesis of Kb-length mRNA.
Enzymatic Ligation Using Splint Oligonucleotides
Enzymatic ligation using splint oligonucleotides is a precise and efficient method for assembling kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides. This method involves using short complementary DNA oligonucleotides, known as splints, to facilitate the alignment and ligation of adjacent RNA oligonucleotides. Here is a detailed breakdown of the process:
Design and Synthesis
Designing RNA Oligonucleotides:
Length and Overlap: RNA oligonucleotides are typically designed to be around 70 nucleotides in length, with 20-30 nucleotide overlaps with adjacent oligonucleotides. These overlapping regions ensure that the oligonucleotides can anneal correctly, forming a continuous RNA sequence.
Sequence Design: The sequences are carefully chosen to minimize secondary structures and ensure that the overlaps are sufficiently stable for effective annealing and ligation.
Synthesis of RNA Oligonucleotides:
Chemical Synthesis: The RNA oligonucleotides are synthesized using automated synthesizers based on phosphoramidite chemistry, which allows for precise control over the sequence and modifications.
Quality Control: Post-synthesis, the oligonucleotides are purified using high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to ensure high purity and correct length.
Designing Splint Oligonucleotides:
Complementary DNA Splints: Splint oligonucleotides are short DNA sequences designed to be complementary to the junctions between adjacent RNA oligonucleotides. They help align the RNA fragments for efficient ligation.
Length: Splints are usually 15-20 nucleotides long, covering the overlap region and extending slightly into the adjacent RNA sequences for stability.
Annealing
Mixing RNA and Splint Oligonucleotides:
Equimolar Ratios: The RNA oligonucleotides and splint oligonucleotides are mixed in equimolar amounts to ensure that each RNA fragment can find its complementary splint.
Annealing Buffer: The mixture is prepared in an annealing buffer typically containing Tris-HCl (to maintain pH), NaCl (to stabilize hybridization), and sometimes MgCl_2 (to stabilize RNA-DNA hybrids).
Annealing Procedure:
Heat Denaturation: The mixture is heated to 95°C for 2 minutes to denature any secondary structures in the RNA.
Gradual Cooling: The mixture is then gradually cooled to room temperature over several hours, allowing the RNA and DNA splints to anneal, forming stable RNA-DNA hybrids.
Ligation
Ligation Reaction Setup:
T4 RNA Ligase: T4 RNA ligase is used to catalyze the formation of phosphodiester bonds between the 3’-hydroxyl and 5’-phosphate ends of adjacent RNA oligonucleotides. This enzyme requires ATP as a cofactor.
Reaction Components: The ligation reaction includes the annealed RNA-splint mixture, T4 RNA ligase, ATP, and a suitable buffer (e.g., 50 mM Tris-HCl, 10 mM MgCl_2, 10 mM DTT).
Optimizing Reaction Conditions:
Temperature: The reaction is typically carried out at 16°C overnight. This lower temperature helps maintain the stability of the RNA-DNA hybrids while allowing efficient ligation.
Duration: Extended incubation times (e.g., overnight) are used to maximize the yield of full-length RNA.
Ligation Efficiency:
Enzyme Concentration: The concentration of T4 RNA ligase and the RNA oligonucleotides are optimized to ensure high efficiency and yield.
Buffer Composition: The buffer conditions are optimized for the activity of T4 RNA ligase, ensuring proper pH and ionic strength.
Removal of Splints
DNase Treatment:
DNase I: After ligation, DNase I is used to digest the DNA splints. DNase I specifically degrades DNA without affecting RNA, ensuring that only the splint oligonucleotides are removed.
Reaction Conditions: The DNase treatment is typically carried out at 37°C for 30 minutes to 1 hour in a buffer containing MgCl_2, which is essential for DNase I activity.
Purification
Purification of Ligated RNA:
Denaturing PAGE: The ligated RNA is purified using denaturing polyacrylamide gel electrophoresis (PAGE). The denaturing conditions (using urea) ensure that the RNA is separated based on size, without secondary structure interference.
Visualization: RNA bands are visualized using UV shadowing or staining with dyes such as ethidium bromide or SYBR Green.
Extraction: The band corresponding to the full-length RNA is excised from the gel, and the RNA is extracted using a gel extraction kit or by eluting in an appropriate buffer.
Alternative Purification Methods:
Column Chromatography: Anion-exchange or affinity chromatography can also be used to purify the RNA. These methods use columns with resins that selectively bind RNA, allowing for the elution of the full-length product.
Elution: The RNA is eluted from the column using a gradient of increasing ionic strength or specific elution buffers designed to release the bound RNA.
Example Protocol for Enzymatic Ligation Using Splint Oligonucleotides
Materials and Reagents:
Synthetic RNA oligonucleotides (70mers) with designed overlaps
DNA splint oligonucleotides (15-20 nucleotides)
T4 RNA ligase
ATP (100 mM stock solution)
Ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl_2, 10 mM DTT)
DNase I
DNase buffer (10 mM Tris-HCl, 2.5 mM MgCl_2, 0.5 mM CaCl_2, pH 7.6)
Denaturing PAGE apparatus and reagents
Gel extraction kit or appropriate elution buffers
Ethanol and salts for RNA precipitation
RNase-free water or TE buffer
Procedure:
Mix RNA and Splint Oligonucleotides:
Combine equimolar amounts of RNA oligonucleotides and DNA splints in an annealing buffer.
Heat to 95°C for 2 minutes to denature secondary structures, then gradually cool to room temperature to allow annealing.
Set Up the Ligation Reaction:
In a new tube, combine the annealed RNA-splint mixture, T4 RNA ligase, ATP, and ligation buffer.
Incubate at 16°C overnight to allow for efficient ligation.
Remove Splint Oligonucleotides:
Add DNase I and DNase buffer to the ligation mixture.
Incubate at 37°C for 30 minutes to 1 hour to digest the DNA splints.
Purify Ligated RNA:
Run the ligation mixture on a denaturing polyacrylamide gel (8-10% polyacrylamide with 7 M urea).
Visualize and excise the band corresponding to the full-length RNA.
Extract the RNA from the gel using a gel extraction kit or by eluting in an appropriate buffer.
Final Purification:
Precipitate the RNA by adding 2.5 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2).
Incubate at -20°C for at least 30 minutes, then centrifuge to pellet the RNA.
Wash the pellet with 70% ethanol, air dry, and resuspend in RNase-free water or TE buffer.
Enzymatic ligation using splint oligonucleotides is a precise method for assembling long RNA sequences from shorter fragments. By leveraging the complementary nature of splint oligonucleotides to align RNA fragments, and using T4 RNA ligase to catalyze their ligation, researchers can produce high-quality, full-length mRNA. This method's detailed steps, including careful design, annealing, ligation, and purification, ensure the successful synthesis of Kb-length mRNA, enabling advanced studies and applications in molecular biology and biotechnology.
Recursive Ligation
Recursive ligation is a method used to assemble kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides by progressively ligating them in pairs to form longer fragments. This process is repeated in multiple steps until the desired full-length RNA is obtained. Each ligation step is followed by purification to ensure high efficiency and yield. Below is a detailed breakdown of the recursive ligation process.
Initial Ligation
Design and Synthesis of RNA Oligonucleotides:
Length and Overlap: RNA oligonucleotides are designed to be around 70 nucleotides in length, with overlapping regions of 20-30 nucleotides. This overlap ensures that the oligonucleotides can anneal correctly, forming a continuous RNA sequence.
Sequence Design: Careful design is necessary to minimize secondary structures and ensure efficient annealing and ligation.
Synthesis of RNA Oligonucleotides:
Chemical Synthesis: RNA oligonucleotides are synthesized using automated synthesizers based on phosphoramidite chemistry.
Quality Control: Purification is carried out using high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to ensure high purity and correct length.
Annealing:
Mixing RNA Oligonucleotides: Combine equimolar amounts of RNA oligonucleotides in an annealing buffer (e.g., 10 mM Tris-HCl, pH 7.5, 50 mM NaCl).
Heat Denaturation: Heat the mixture to 95°C for 2 minutes to denature any secondary structures, then gradually cool to room temperature over several hours to allow annealing.
Ligation:
T4 RNA Ligase: T4 RNA ligase catalyzes the formation of phosphodiester bonds between the 3’-hydroxyl and 5’-phosphate ends of adjacent RNA oligonucleotides.
Reaction Setup: Combine the annealed RNA oligonucleotides with T4 RNA ligase, ATP, and ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl_2, 10 mM DTT).
Incubation: Incubate the reaction at 16°C overnight to ensure efficient ligation.
Recursive Steps
Purification After Each Ligation:
Denaturing PAGE: Purify the ligated RNA using denaturing polyacrylamide gel electrophoresis (PAGE) to separate the ligated products from unligated oligonucleotides.
Visualization and Extraction: Visualize RNA bands using UV shadowing or staining, excise the desired band, and extract the RNA using a gel extraction kit.
Combining Fragments:
Pairwise Ligation: Pair the ligated RNA fragments in subsequent steps, continuing to combine them into longer sequences. For example, if the initial ligation step combined 70mer oligonucleotides into 140mer fragments, the next step would combine these 140mer fragments into 280mer sequences.
Equimolar Mixing: Ensure that the RNA fragments are mixed in equimolar ratios to promote efficient annealing and ligation.
Annealing and Ligation:
Annealing: Heat the RNA mixture to 95°C for 2 minutes, then gradually cool to room temperature to allow annealing.
Ligation: Use T4 RNA ligase, ATP, and ligation buffer to catalyze the formation of phosphodiester bonds between the RNA fragments. Incubate at 16°C overnight.
Iteration:
Repeat Steps: Continue the process of purification, combining, annealing, and ligation in recursive steps until the desired Kb-length RNA is achieved. Each step approximately doubles the length of the RNA fragments until the full-length RNA is obtained.
Final Purification
Denaturing PAGE:
Final Gel Purification: Once the desired full-length RNA is obtained, run the ligated product on a denaturing polyacrylamide gel to separate it from any remaining shorter fragments.
Visualization and Extraction: Visualize the RNA bands, excise the full-length RNA band, and extract the RNA using a gel extraction kit or elution buffer.
Ethanol Precipitation:
Precipitation: Add 2.5 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2) to the RNA solution. Mix and incubate at -20°C for at least 30 minutes to precipitate the RNA.
Centrifugation: Centrifuge at high speed (e.g., 12,000 x g) for 15 minutes at 4°C to pellet the RNA.
Washing: Wash the RNA pellet with 70% ethanol to remove any residual salts and contaminants. Centrifuge again to pellet the RNA.
Resuspension: Air dry the RNA pellet and resuspend in RNase-free water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).
Example Protocol for Recursive Ligation
Materials and Reagents:
Synthetic RNA oligonucleotides (70mers) with designed overlaps
T4 RNA ligase
ATP (100 mM stock solution)
Ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl_2, 10 mM DTT)
Denaturing PAGE apparatus and reagents
Gel extraction kit or appropriate elution buffers
Ethanol and sodium acetate for RNA precipitation
RNase-free water or TE buffer
Procedure:
Initial Ligation:
Combine equimolar amounts of RNA oligonucleotides in an annealing buffer.
Heat to 95°C for 2 minutes, then gradually cool to room temperature to allow annealing.
Set up the ligation reaction with annealed RNA, T4 RNA ligase, ATP, and ligation buffer.
Incubate at 16°C overnight for efficient ligation.
Purify Ligated RNA:
Run the ligation mixture on a denaturing polyacrylamide gel.
Visualize and excise the band corresponding to the ligated RNA.
Extract the RNA from the gel using a gel extraction kit.
Recursive Ligation:
Combine equimolar amounts of the ligated RNA fragments from the initial ligation step.
Heat to 95°C for 2 minutes, then gradually cool to room temperature to allow annealing.
Set up the ligation reaction with annealed RNA fragments, T4 RNA ligase, ATP, and ligation buffer.
Incubate at 16°C overnight.
Repeat the purification and ligation steps, progressively combining longer RNA fragments until the desired Kb-length RNA is obtained.
Final Purification:
Run the final ligation mixture on a denaturing polyacrylamide gel.
Visualize and excise the full-length RNA band.
Extract the RNA from the gel using a gel extraction kit.
Precipitate the RNA with ethanol and sodium acetate, centrifuge to pellet the RNA, wash with 70% ethanol, and resuspend in RNase-free water or TE buffer.
Recursive ligation is an effective method for assembling long RNA sequences from shorter oligonucleotides. By iteratively ligating and purifying RNA fragments, researchers can produce high-quality, full-length mRNA suitable for various applications. The detailed steps outlined above highlight the importance of careful design, annealing, ligation, and purification in achieving successful Kb-length RNA synthesis. This method's precision and efficiency make it a valuable tool in molecular biology and biotechnology.
Chemical Ligation
Chemical ligation is a method used to assemble kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides through the use of specific chemical reactions. This method involves the incorporation of reactive groups at the ends of RNA oligonucleotides, which facilitate the formation of covalent bonds without the need for enzymes. Below is a detailed breakdown of the chemical ligation process.
Design and Synthesis
Designing RNA Oligonucleotides:
Length and Overlap: RNA oligonucleotides are typically designed to be around 70 nucleotides in length, with 20-30 nucleotide overlaps to ensure proper alignment and hybridization.
Sequence Design: Careful design is required to minimize secondary structures and ensure that the reactive groups are accessible for ligation.
Synthesis of RNA Oligonucleotides:
Chemical Synthesis: RNA oligonucleotides are synthesized using automated synthesizers based on phosphoramidite chemistry, allowing precise control over sequence and modifications.
Reactive Group Incorporation: During synthesis, specific chemical groups are added to the 3’- and 5’-ends of the oligonucleotides. Common modifications include:
3’-Amino Group: Facilitates the formation of a covalent bond with a complementary reactive group.
5’-Phosphate Group: Activates the 5’-end for ligation.
Quality Control:
Purification: Post-synthesis, oligonucleotides are purified using high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to ensure high purity and correct length.
Verification: The presence of reactive groups and correct sequences are verified using mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy.
Annealing
Mixing RNA Oligonucleotides:
Equimolar Ratios: RNA oligonucleotides are mixed in equimolar amounts to ensure each fragment has an equal chance of finding its complementary partner.
Annealing Buffer: The mixture is prepared in an annealing buffer containing Tris-HCl, NaCl, and sometimes MgCl_2 to stabilize hybridization.
Annealing Procedure:
Heat Denaturation: Heat the mixture to 95°C for 2 minutes to denature any secondary structures.
Gradual Cooling: Gradually cool the mixture to room temperature over several hours to allow complementary regions to anneal, forming stable RNA-RNA duplexes.
Chemical Ligation Reaction
Setting Up the Ligation Reaction:
Reactants: Combine the annealed RNA oligonucleotides with the necessary chemical reagents to facilitate ligation. Typical reagents include:
Carbodiimides (e.g., EDC, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide): Facilitate the formation of covalent bonds between the 3’-amino and 5’-phosphate groups.
Catalysts or Activators: Additional chemicals may be used to enhance the reaction efficiency.
Optimizing Reaction Conditions:
Temperature and pH: The reaction is typically carried out at room temperature or slightly elevated temperatures (25-37°C) to balance reaction rate and stability. The pH is usually maintained around 7.0 to 8.0.
Reaction Time: The reaction is allowed to proceed for several hours to overnight, depending on the efficiency of the chemical reagents used.
Reaction Efficiency:
Concentration: High concentrations of RNA oligonucleotides and reagents are used to drive the ligation reaction forward.
Buffer Composition: The buffer is optimized for the chemical reagents used, ensuring proper ionic strength and pH.
Purification
Purification of Ligated RNA:
Denaturing PAGE: The ligated RNA is purified using denaturing polyacrylamide gel electrophoresis (PAGE). Denaturing conditions (using urea) ensure separation based on size.
Visualization: RNA bands are visualized using UV shadowing or staining with dyes such as ethidium bromide or SYBR Green.
Extraction: The band corresponding to the full-length RNA is excised from the gel, and the RNA is extracted using a gel extraction kit or by eluting in an appropriate buffer.
Alternative Purification Methods:
Column Chromatography: Anion-exchange or affinity chromatography can be used to purify the RNA. These methods use columns with resins that selectively bind RNA, allowing for the elution of the full-length product.
Elution: The RNA is eluted from the column using a gradient of increasing ionic strength or specific elution buffers designed to release the bound RNA.
Example Protocol for Chemical Ligation
Materials and Reagents:
Synthetic RNA oligonucleotides (70mers) with designed overlaps and reactive groups (3’-amino and 5’-phosphate)
Chemical reagents (e.g., EDC)
Annealing buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl)
Ligation buffer (optimized for the specific chemical reagents used)
Denaturing PAGE apparatus and reagents
Gel extraction kit or appropriate elution buffers
Ethanol and sodium acetate for RNA precipitation
RNase-free water or TE buffer
Procedure:
Mix and Anneal RNA Oligonucleotides:
Combine equimolar amounts of RNA oligonucleotides in an annealing buffer.
Heat to 95°C for 2 minutes, then gradually cool to room temperature to allow annealing.
Set Up the Chemical Ligation Reaction:
In a new tube, combine the annealed RNA oligonucleotides with the chemical reagents (e.g., EDC) and ligation buffer.
Incubate at room temperature or 25-37°C for several hours to overnight, ensuring optimal reaction conditions.
Purify Ligated RNA:
Run the ligation mixture on a denaturing polyacrylamide gel (8-10% polyacrylamide with 7 M urea).
Visualize and excise the band corresponding to the full-length RNA.
Extract the RNA from the gel using a gel extraction kit or by eluting in an appropriate buffer.
Final Purification:
Precipitate the RNA by adding 2.5 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2).
Incubate at -20°C for at least 30 minutes, then centrifuge to pellet the RNA.
Wash the pellet with 70% ethanol, air dry, and resuspend in RNase-free water or TE buffer.
Chemical ligation offers a unique approach to assembling long RNA sequences from shorter oligonucleotides by leveraging specific chemical reactions to form covalent bonds. This method bypasses the need for enzymatic ligation, making it suitable for scenarios where enzymatic activity may be hindered or undesirable. By carefully designing the RNA sequences, optimizing the reaction conditions, and employing precise purification techniques, researchers can produce high-quality, full-length mRNA suitable for various applications in molecular biology and biotechnology. The detailed steps outlined above provide a comprehensive guide to successfully implementing chemical ligation for RNA assembly.
Hybridization-Based Assembly Using Overlapping Oligonucleotides
Hybridization-based assembly is a method for constructing kilobase-length (Kb-length) mRNA from shorter RNA oligonucleotides through precise annealing and ligation. This method relies on the hybridization of complementary overlapping regions of RNA oligonucleotides to form a continuous RNA strand, which is then ligated to produce full-length mRNA. Below is a detailed breakdown of each step involved in this process.
Design and Synthesis
Designing RNA Oligonucleotides:
Length and Overlap: RNA oligonucleotides are typically designed to be around 70 nucleotides in length, with 20-30 nucleotide overlaps with adjacent oligonucleotides. These overlaps ensure that the oligonucleotides can hybridize correctly, forming a continuous RNA sequence.
Sequence Design: Sequences are designed to minimize secondary structures and ensure that overlapping regions are stable enough to hybridize effectively.
Synthesis of RNA Oligonucleotides:
Chemical Synthesis: RNA oligonucleotides are synthesized using automated synthesizers based on phosphoramidite chemistry, allowing precise control over the sequence and modifications.
Quality Control: After synthesis, the oligonucleotides are purified using high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) to ensure high purity and correct length.
Hybridization
Mixing RNA Oligonucleotides:
Equimolar Ratios: RNA oligonucleotides are mixed in equimolar amounts to ensure that each fragment has an equal chance of finding its complementary overlap.
Annealing Buffer: The mixture is prepared in an annealing buffer typically containing Tris-HCl (to maintain pH), NaCl (to stabilize hybridization), and sometimes MgCl_2 (to stabilize RNA-RNA interactions).
Hybridization Procedure:
Heat Denaturation: The mixture is heated to 95°C for 2 minutes to denature any secondary structures in the RNA.
Gradual Cooling: The mixture is then gradually cooled to room temperature over several hours, allowing the complementary regions of the RNA oligonucleotides to hybridize and form stable RNA duplexes.
Ligation
Setting Up the Ligation Reaction:
T4 RNA Ligase: T4 RNA ligase is used to catalyze the formation of phosphodiester bonds between the 3’-hydroxyl and 5’-phosphate ends of adjacent RNA oligonucleotides.
Reaction Components: The ligation reaction includes the hybridized RNA oligonucleotides, T4 RNA ligase, ATP, and a suitable ligation buffer (e.g., 50 mM Tris-HCl, 10 mM MgCl_2, 10 mM DTT).
Optimizing Reaction Conditions:
Temperature: The ligation reaction is typically carried out at a low temperature (around 16°C) to stabilize the RNA duplexes and minimize the formation of secondary structures that could hinder ligation.
Duration: The reaction is allowed to proceed overnight to maximize the yield of full-length RNA.
Ligation Efficiency:
Enzyme Concentration: The concentration of T4 RNA ligase and the RNA oligonucleotides are optimized to ensure high efficiency and yield.
Buffer Composition: The buffer conditions are optimized for the activity of T4 RNA ligase, ensuring proper pH and ionic strength.
Purification
Purification of Ligated RNA:
Denaturing PAGE: The ligated RNA is purified using denaturing polyacrylamide gel electrophoresis (PAGE). Denaturing conditions (using urea) ensure that the RNA is separated based on size, without secondary structure interference.
Visualization: RNA bands are visualized using UV shadowing or staining with dyes such as ethidium bromide or SYBR Green.
Extraction: The band corresponding to the full-length RNA is excised from the gel, and the RNA is extracted using a gel extraction kit or by eluting in an appropriate buffer.
Alternative Purification Methods:
Column Chromatography: Anion-exchange or affinity chromatography can be used to purify the RNA. These methods use columns with resins that selectively bind RNA, allowing for the elution of the full-length product.
Elution: The RNA is eluted from the column using a gradient of increasing ionic strength or specific elution buffers designed to release the bound RNA.
Example Protocol for Hybridization-Based Assembly
Materials and Reagents:
Synthetic RNA oligonucleotides (70mers) with designed overlaps
T4 RNA ligase
ATP (100 mM stock solution)
Ligation buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl_2, 10 mM DTT)
Denaturing PAGE apparatus and reagents
Gel extraction kit or appropriate elution buffers
Ethanol and sodium acetate for RNA precipitation
RNase-free water or TE buffer
Procedure:
Mix and Anneal RNA Oligonucleotides:
Combine equimolar amounts of RNA oligonucleotides in an annealing buffer.
Heat the mixture to 95°C for 2 minutes to denature secondary structures.
Gradually cool the mixture to room temperature over several hours to allow hybridization.
Set Up the Ligation Reaction:
In a new tube, combine the hybridized RNA oligonucleotides, T4 RNA ligase, ATP, and ligation buffer.
Incubate at 16°C overnight to ensure efficient ligation.
Purify Ligated RNA:
Run the ligation mixture on a denaturing polyacrylamide gel (8-10% polyacrylamide with 7 M urea).
Visualize and excise the band corresponding to the full-length RNA.
Extract the RNA from the gel using a gel extraction kit or by eluting in an appropriate buffer.
Final Purification:
Precipitate the RNA by adding 2.5 volumes of ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2).
Incubate at -20°C for at least 30 minutes, then centrifuge to pellet the RNA.
Wash the pellet with 70% ethanol, air dry, and resuspend in RNase-free water or TE buffer.
Hybridization-based assembly using overlapping oligonucleotides is a precise and efficient method for synthesizing long RNA sequences. By leveraging the inherent base-pairing properties of RNA, this method allows for the accurate assembly of Kb-length mRNA. The detailed steps outlined above highlight the importance of careful design, annealing, ligation, and purification in achieving successful RNA assembly. This method's versatility and reliability make it a valuable tool in molecular biology and biotechnology.
Nucleobase and Backbone Modifications for mRNA Translation Studies
Incorporating modifications into RNA sequences can significantly enhance their stability, functionality, and detectability in vivo. Here are various nucleobase and backbone modifications that can be used for in vivo studies of tagged mRNA translation:
Nucleobase Modifications
6-Methylisoxanthopterin (6-MI): A fluorescent analog of guanine, 6-MI can be incorporated into RNA to track its localization and translation in real-time.
2-Aminopurine (2-AP): This fluorescent analog of adenine is used to study RNA dynamics and interactions.
Pseudouridine (Ψ): Enhancing RNA stability and reducing immune recognition, pseudouridine is beneficial for in vivo applications.
N^6-Methyladenosine (m^6A): Influences RNA metabolism and translation efficiency, useful for studying mRNA regulation in vivo.
N^1-Methylpseudouridine (m^1Ψ): Similar to pseudouridine but with an additional methyl group, m^1Ψ increases RNA stability and translation efficiency while reducing immunogenicity.
5-Methylcytidine (5mC): A methylated form of cytidine that impacts RNA stability and interactions with RNA-binding proteins.
5-Hydroxymethylcytidine (5hmC): Influences RNA metabolism and is less prone to deamination than cytidine.
5-Fluorouridine (5FU): A fluorinated analog of uridine used to study RNA processing and degradation.
Inosine (I): An adenine analog that pairs with cytosine, increasing wobble pairing and flexibility in RNA interactions.
Backbone Modifications
2’-O-Methyl (2’-OMe): Adding a methyl group to the 2’-hydroxyl of the ribose sugar increases RNA stability and resistance to nuclease degradation.
Phosphorothioate (PS) Linkages: Substituting one of the non-bridging oxygen atoms in the phosphate backbone with a sulfur atom enhances RNA resistance to nucleases.
2’-Fluoro (2’-F) Nucleotides: Fluorine substitution at the 2’-position of the ribose increases RNA stability against enzymatic degradation.
2’-O-Methoxyethyl (2’-MOE): Adds a methoxyethyl group to the 2’ position, increasing RNA stability and affinity for complementary strands.
Phosphoramidate Linkages: Modifications to the phosphate backbone that increase resistance to nuclease degradation and enhance stability.
Morpholino Oligomers: Synthetic oligonucleotides with morpholine rings and phosphorodiamidate linkages, highly stable and resistant to enzymatic degradation.
L-RNA (Spiegelmers): Mirror-image RNA oligonucleotides that are not recognized by nucleases, offering high stability in biological environments.
Conclusion
Advances in nucleobase and backbone modifications, along with sophisticated assembly techniques, have significantly improved the utility and performance of mRNA for various applications. These modifications enhance mRNA stability, translation efficiency, and in vivo detectability, making them indispensable tools in the field of molecular biology. The methods for assembling Kb-length mRNA from shorter oligonucleotides are diverse, each offering unique advantages depending on the desired outcome. As research continues to evolve, these techniques will likely become even more refined, further expanding the potential of mRNA-based technologies in medicine and research.