In recent years, the role of RNA molecules in cellular processes and human disorders has become increasingly evident. From regulating gene expression to serving as catalytic agents, RNA's versatility stems from its structural complexity. Despite being composed of only four nucleotides, RNA can fold into intricate secondary and tertiary structures crucial for its function. Understanding RNA function necessitates deciphering its spatial structure, a task accomplished through techniques like crystallography, nuclear magnetic resonance (NMR), and cryogenic electron microscopy (cryo-EM). However, these methods often demand substantial sample quantities. To address this challenge, large-scale synthesis of RNA is imperative. Chemical synthesis and in vitro transcription are the two primary approaches employed for this purpose, each with its own advantages and limitations.
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Introduction
RNA, a pivotal player in cell biology, has emerged as a multifaceted molecule with diverse functions, ranging from gene regulation to disease pathogenesis. Initially indistinguishable from DNA, RNA's unique properties were gradually unveiled through pioneering research efforts. Today, RNA serves as a crucial tool in biotechnology and medicine, exemplified by the mRNA-based vaccines combatting the COVID-19 pandemic.
Understanding RNA's three-dimensional structure is essential for elucidating its functions and dynamics, as well as for drug development. Pioneering studies, such as the discovery of the double helix structure of DNA by Watson and Crick, laid the foundation for structural studies of RNA. Subsequent advancements led to the elucidation of various RNA structures, shedding light on their functional principles and interactions.
Methods of RNA Synthesis
RNA synthesis can be accomplished through purification from biological sources, chemical synthesis, or enzymatic synthesis via in vitro transcription. While purification from cells is suitable for large, highly abundant RNAs, chemical synthesis and in vitro transcription offer more universal approaches for synthesizing RNA molecules, particularly for structural studies.
Chemical Synthesis
Chemical synthesis, pioneered by Bruce Merrifield, revolutionized the production of nucleic acids like DNA and RNA. This method employs solid-phase synthesis on materials like controlled-pore glass or crosslinked polystyrene, utilizing phosphoramidites adorned with various protective groups to control reactivity during synthesis. Protection groups such as TOM and TBDMS shield the 2'-hydroxyl, while DMT blocks the 5' OH group, crucial for RNA synthesis efficiency. Recent advancements with potent activators like ETT and BTT have expedited coupling steps.
The process unfolds in cycles, each comprising detritylation, coupling, capping, and oxidation steps. Despite high individual coupling yields, the overall efficiency diminishes progressively with cycle count, especially for longer oligomers. For instance, a 25 nt-long oligomer synthesis can yield about 79%, assuming 99% single coupling efficiency, but drops to approximately 48% if coupling efficiency dips to 97%. Longer oligomers face even lower yields, rendering chemical synthesis impractical without special considerations, like introducing modified nucleotides.
Fig 1. Overview of the solid-phase chemical RNA synthesis cycle (Ryczek M., et al. 2022).
Nonetheless, chemical synthesis offers advantages like easy incorporation of modifications and less sequence constraints compared to in vitro transcription. For longer sequences, strategies like split–join ligation can be employed, maintaining the benefits of chemical synthesis while overcoming length limitations. Thus, despite its limitations, chemical synthesis remains a powerful tool for nucleic acid production, especially when coupled with innovative strategies to enhance efficiency and versatility.
In Vitro RNA Synthesis Using T7 RNA Polymerase
In vitro RNA synthesis using T7 RNA polymerase is a common technique for generating RNA molecules. It involves employing a DNA template containing a T7 RNA polymerase promoter sequence, followed by the sequence encoding the desired RNA molecule. The polymerase binds to this template at the promoter region and initiates RNA synthesis. Elongation of the RNA chain ceases when the enzyme dissociates from the 3'-end of the DNA template. While in vitro synthesis can yield milligrams of RNA, the efficiency depends on various factors, with the nucleotide composition downstream of the T7 promoter being crucial. An optimal start typically involves one or more consecutive guanosine residues.
However, a notable issue with in vitro RNA synthesis is the production of short, abortive transcripts and transcripts with extra nucleotides added to the 3'-end due to polymerase slippage. Separating these products from full-length transcripts is relatively straightforward for short fragments but challenging for those with a few extra nucleotides. This heterogeneity reduces the overall yield and crystallization potential of the RNA sample. Additionally, the presence of a tri-phosphate group at the 5'-end of the RNA, resulting from the transcription process, can hinder crystallization. To address this, converting the triphosphate to a hydroxyl group may be necessary, which can be achieved using guanosine residues or alkaline phosphatase during transcription. Furthermore, in vitro transcription can be used to produce capped RNAs, which are vital for various cellular processes, albeit with limitations in efficiency and structure diversity. Recent advancements aim to overcome these limitations, expanding the repertoire of available cap structures and increasing incorporation efficiency.
Ribozymes for RNA Production with Homogeneous Ends
Ribozymes, catalytic RNA molecules, offer an attractive solution for generating RNA with precise 5' and 3' ends. Hammerhead, hairpin, hepatitis delta virus (HDV), and Varkud satellite (VS) ribozymes are among the most commonly used ribozymes for this purpose. These ribozymes catalyze site-specific cleavage of RNA, enabling the production of RNA molecules with defined ends.
Preparation of RNA with Homogeneous 3' End Using Modified DNA Template
Modification of DNA templates with C2'-methoxy groups represents an alternative strategy for reducing RNA 3' end heterogeneity during in vitro transcription. This modification mitigates non-templated nucleotide additions by RNA polymerase, resulting in the production of RNA with homogeneous 3' ends. Additionally, modification of DNA templates allows for the synthesis of RNA with specific 5' end modifications, such as monophosphate groups.
In conclusion, the synthesis of RNA molecules for structural studies represents a multifaceted endeavor, drawing upon diverse methodologies and optimization strategies. Chemical synthesis and in vitro transcription each offer unique advantages and limitations, which must be carefully considered in the context of specific RNA constructs and research goals. Incorporation of ribozymes and modification of DNA templates represent powerful approaches for enhancing the homogeneity and quality of synthesized RNA molecules, paving the way for further insights into the structure and function of RNA in cellular processes and disease pathogenesis.
References
- Ryczek M., et al. Overview of methods for large-scale RNA synthesis. Applied Sciences. 2022, 12(3): 1543.
- Merrifield R.B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 1963, 85(14): 2149-54.
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