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What is the core mechanism of siRNA mediated RNA interference (RNAi)?
1. Generation of Mature siRNA (Double-Stranded RNA Processing)
siRNA originates from the cleavage of exogenous or endogenous double-stranded RNA (dsRNA) by the Dicer enzyme (a member of the RNase III family):
Source of dsRNA: Exogenous sources include viral dsRNA (e.g., from RNA viruses) or artificially synthesized dsRNA (e.g., siRNA used in experiments); endogenous sources are rare (mostly from transposons or repeat sequences).
Cleavage process: Dicer recognizes dsRNA and cleaves it into short dsRNA fragments (21–23 nucleotides in length) with two key structural features: a 5′-phosphate group and a 2-nucleotide overhang at the 3′-end—these structures are essential for subsequent RISC assembly.
2. Assembly of the RNA-Induced Silencing Complex (RISC)
The mature siRNA duplex binds to the Argonaute (Ago) protein (the core catalytic component of RISC) and other accessory proteins (e.g., RNA helicase) to form the active RISC, with two critical sub-steps:
Strand separation: Under the action of RNA helicase, the siRNA duplex is unwound into two single strands:
Guide strand: The strand with higher stability at the 5′-end (determined by base pairing energy); it remains bound to Ago and serves as the "guide" for target recognition.
Passenger strand: The complementary strand of the guide strand; it is structurally unstable and is degraded by Ago’s nuclease activity, ensuring RISC only retains the functional guide strand.
Activation of RISC: After strand separation, the Ago-guide strand complex (active RISC) is formed, and its "seed region" (nucleotides 2–8 of the guide strand) is exposed to prepare for target mRNA recognition.
3. Target mRNA Recognition and Post-Transcriptional Gene Silencing (PTGS)
The core function of active RISC is to specifically silence target gene expression by recognizing and degrading mRNA, relying on base complementarity between the guide strand and target mRNA:
Specific recognition: The guide strand of RISC binds to the 3′-untranslated region (3′-UTR) or coding region of target mRNA through complete base complementarity (a key feature distinguishing siRNA from miRNA, which usually binds via incomplete complementarity).
mRNA cleavage and degradation: The Ago protein in RISC has a conserved PIWI domain (a nuclease domain) that cleaves the phosphodiester bond of the target mRNA at the site 10–11 nucleotides downstream of the guide strand’s 5′-end. The cleaved mRNA fragments are further degraded by cellular exonucleases (e.g., XRN1), preventing them from being translated into proteins.
Result of silencing: Since the target mRNA is degraded before translation, the expression of the corresponding target gene is significantly reduced (transcription is unaffected; silencing occurs at the post-transcriptional level).
Key Summary
The core mechanism of siRNA-mediated RNAi can be simplified as: dsRNA cleavage by Dicer → active RISC assembly (guide strand retention) → target mRNA cleavage via RISC → post-transcriptional gene silencing. This process is highly specific (dependent on guide strand-mRNA complementarity) and efficient (one RISC can cleave multiple mRNA molecules), making it a core tool for gene function research.
Why must a sequencing primer bind specifically to the DNA template?
The key to successful sequencing is that the sequencing primer has only one binding site on the DNA template molecule. If the sequencing primer has more than one binding site on the DNA template, it will cause the primer to initiate extension at multiple sites during the sequencing reaction. This will manifest as double peaks or messy peaks on the sequencing chromatogram, making the sequence unreadable.