Mechanism of dsRNA Formation and Its Immunogenicity in In Vitro Transcription Reactions
Overview of RNA Synthesis Technology
The development of modern RNA therapies relies on efficient and reliable RNA synthesis technologies. Currently, RNA synthesis is mainly divided into two major technical routes: chemical synthesis and enzymatic synthesis. Chemical synthesis has significant advantages in the preparation of short-chain RNAs (such as siRNAs, aptamers, etc.), allowing precise control over the connection order of each nucleotide through solid-phase synthesis technology. However, as the length of the RNA chain increases, the efficiency of chemical synthesis decreases exponentially due to cumulative losses at each step's reaction yield. When RNA lengths exceed 100-150 nucleotides, the actual yield from chemical methods often fails to meet therapeutic needs.
In contrast, enzymatic synthesis can overcome length limitations and is suitable for preparing various lengths of RNAs. Among them, T7 RNA polymerase has become the most commonly used tool enzyme due to its high efficiency and stability during transcription reactions. This enzyme is easy to express recombinantly in E.coli systems with high processing capabilities that allow it to efficiently synthesize long RNA molecules up to several thousand nucleotides during in vitro transcription reactions. However, products synthesized by this method often exhibit higher immunogenicity which poses a significant bottleneck for clinical applications; this immunogenicity arises both from structural features inherent to target RNAs themselves as well as various by-products generated during transcription processes. A deep understanding of these sources and mechanisms behind immunogenicity is crucial for developing safe and effective RNA therapeutic products.
Mechanism of Immune Recognition for dsRNA
In eukaryotic cells, RIG-I-like receptors (RLRs) are key pattern recognition receptors that identify viral RNAs; among them RIG-I and MDA5 serve as two primary intracellular double-stranded RNA (dsRNA) sensors capable specifically recognizing exogenous viral RNAs while activating antiviral immune responses—though they share similar functions their recognition mechanisms differ significantly.
RIG-I’s protein structure consists three functional domains: an N-terminal tandem pair CARD domains (Caspase Activation Recruitment Domains), a central helicase domain with ATP hydrolysis activity along with a C-terminal domain (CTD). The CTD recognizes either triphosphate groups or diphosphate groups at 5' ends while also requiring interaction between unmodified 2'-OH group present on first nucleotide within an mRNA molecule; meanwhile helicase domain binds directly onto double-stranded regions within said mRNAs where its ATPase activity plays critical regulatory roles throughout recognition process itself. When RIG-I stably binds ligands conformational changes relieve self-inhibition between CARD & helicase domains exposing latter enabling interactions downstream connector proteins MAVS thus initiating antiviral signaling pathways.
MDA5’s mechanism diverges entirely relying not upon special structures found at terminal ends but rather internal double stranded regions typically ranging from 0.5-1kb long forming spiral filamentous structures via head-to-tail assembly along dsRNA backbone—a multimerization process allows exterior positioned CARD domains oligomerize subsequently activating downstream MAVS signaling pathway effectively inducing immune response activation too! Notably enough though virus-derived sequences generally display unique characteristics such as phosphate groups located solely at their respective five-prime termini alongside unmodified riboses host derived counterparts avoid revealing these stimulatory traits through modifications like cap1/cap2 structures applied atop those same sites instead!
Sources Of IVT Product Immunogenicity
Traditional views hold that immune reactivity observed amongst IVT products primarily stems from presence associated triphosphate moieties located right there upon their five-prime terminals recognized specifically by RIG-I however experimental evidence indicates even when phosphatases remove such entities substantial levels remain active still suggesting other contributors exist therein! For instance Sun Hur et al published pivotal findings back around year twenty eighteen showcasing four distinct sequence compositions made up comprising totaling five hundred twelve nt-long templates designed ensuring no stable secondary formations occurred whatsoever leading investigators able utilize interferon promoter-driven luciferase reporter assays confirming all resultant transcripts activated both aforementioned signal transduction pathways strongly regardless if treated or untreated using alkaline phosphatases eliminating ppp residues altogether thereby validating hypothesis surrounding additional non-p3p dependent stimuli residing inside original constructs! Further gel electrophoresis analyses reveal complex mixtures produced across multiple runs showing clear bands indicating ssRNAs yielding yellow fluorescence versus green fluorescing corresponding dSRNAs detected below—confirming previous hypotheses concerning dual strand formation potential existing amidst overall populations studied further supported via nuclease digestion experiments performed afterwards proving specificity against individual types being tested successfully distinguishing outcomes based solely off initial designs utilized initially prompting further investigations exploring how best isolate functional components thereafter leading us down paths towards elucidating underlying principles governing production methodologies involved hereafter going forward......