Biologics: RNA interference MCQs With Answer

Biologics: RNA interference (RNAi) MCQs With Answer

This quiz set focuses on RNA interference—its molecular mechanisms, therapeutic applications, delivery strategies, chemical modifications, safety considerations and evaluation methods—designed for M.Pharm students studying Biological Evaluation of Drug Therapy. Questions probe detailed aspects such as Dicer and Argonaute function, siRNA/shRNA/miRNA distinctions, design rules (seed region, 3′ overhangs, thermodynamic asymmetry), delivery platforms (LNP, GalNAc), immunogenicity (TLRs, RIG‑I), clinical examples (patisiran, inclisiran, givosiran), and analytical assays used in preclinical and clinical development. The aim is to deepen understanding of how RNAi drugs are optimized, evaluated and translated into safe, effective therapies.

Q1. Which enzyme cleaves long double‑stranded RNA into ~21–23 nucleotide siRNA duplexes in the canonical RNAi pathway?

• RNA polymerase II
• Dicer
• Argonaute 2
• Ribonuclease H

Correct Answer: Dicer

Q2. In RNAi, which protein is the catalytic component of RISC that mediates target mRNA cleavage when perfect complementarity exists?

• Dicer
• Drosha
• Argonaute 2 (Ago2)
• RNase III

Correct Answer: Argonaute 2 (Ago2)

Q3. What is the typical length and structural feature of a canonical siRNA duplex used for RNAi?

• ~100 nucleotides single‑stranded with 5′ cap
• ~21–23 base pairs with 2‑nucleotide 3′ overhangs
• ~50 base pairs blunt‑ended duplex
• ~8–12 nucleotides microRNA‑like seed only

Correct Answer: ~21–23 base pairs with 2‑nucleotide 3′ overhangs

Q4. Which region of the siRNA guide strand is most critical for off‑target effects via partial complementarity to mRNA 3’UTRs?

• Positions 15–21 (3′ end)
• Central region (positions 9–12)
• Seed region (positions 2–8 from 5′ end)
• Entire strand equally

Correct Answer: Seed region (positions 2–8 from 5′ end)

Q5. Which chemical modification is commonly introduced at the 2′ position of ribose to increase siRNA nuclease stability and reduce immune activation?

• 5′ capping with m7G
• 2′-O‑methyl or 2′-fluoro substitutions
• Polyadenylation at 3′ end
• 5′ phosphorylation only

Correct Answer: 2′-O‑methyl or 2′-fluoro substitutions

Q6. Which delivery strategy uses N‑acetylgalactosamine (GalNAc) conjugation to selectively target hepatocytes via the asialoglycoprotein receptor?

• Lipid nanoparticle (LNP) encapsulation
• Viral vector transduction
• GalNAc‑siRNA conjugation
• Polyethyleneimine (PEI) polyplexes

Correct Answer: GalNAc‑siRNA conjugation

Q7. Which clinically approved siRNA therapeutic is delivered using lipid nanoparticles for treatment of hereditary transthyretin‑mediated amyloidosis?

• Inclisiran (Leqvio)
• Patisiran (Onpattro)
• Givosiran (Givlaari)
• Fomivirsen

Correct Answer: Patisiran (Onpattro)

Q8. What is a primary advantage of using GalNAc conjugation over systemic LNP delivery for liver‑targeted siRNA therapy?

• Enables crossing of the blood‑brain barrier
• Direct receptor‑mediated uptake into hepatocytes and subcutaneous dosing
• Prevents renal clearance completely
• Eliminates need for chemical modifications

Correct Answer: Direct receptor‑mediated uptake into hepatocytes and subcutaneous dosing

Q9. Which innate immune sensors are most commonly implicated in recognizing unmodified single‑stranded RNA and causing immune activation with some siRNA preparations?

• Toll‑like receptors 7/8 and RIG‑I
• Caspase‑3 and caspase‑8
• Complement C3 and C5
• TLR4 and NOD2

Correct Answer: Toll‑like receptors 7/8 and RIG‑I

Q10. Which design principle helps bias guide strand selection during RISC loading to favor one strand over the passenger strand?

• Making both ends of duplex equally thermodynamically stable
• Thermodynamic asymmetry with lower stability at the 5′ end of intended guide
• Adding a 5′ cap to the passenger strand
• Extending duplex length to >50 nt

Correct Answer: Thermodynamic asymmetry with lower stability at the 5′ end of intended guide

Q11. Which analytical assay is commonly used to quantify on‑target mRNA knockdown after siRNA treatment in cells or tissues?

• Western blot for DNA
• qRT‑PCR (quantitative reverse transcription PCR)
• ELISA for genomic DNA
• Mass spectrometry of lipids

Correct Answer: qRT‑PCR (quantitative reverse transcription PCR)

Q12. What toxicology concern is most directly related to chronic accumulation of delivery vehicles (e.g., LNP components) in liver or spleen during repeated siRNA dosing?

• Immediate anaphylactic shock only
• Organ‑specific accumulation leading to hepatotoxicity or splenic changes
• Increased protein translation globally
• Activation of reverse transcriptase activity

Correct Answer: Organ‑specific accumulation leading to hepatotoxicity or splenic changes

Q13. Which of the following chemical backbone modifications increases nuclease resistance but can alter pharmacokinetics and protein binding?

• Phosphodiester linkage
• Phosphorothioate (PS) linkage
• 2′-O‑methylation only
• Unmodified RNA backbone

Correct Answer: Phosphorothioate (PS) linkage

Q14. Which in vitro assay is commonly used to detect incorporation of an siRNA guide into Ago2‑containing RISC complexes?

• Agarose gel electrophoresis of DNA
• RISC immunoprecipitation (RIP) followed by RT‑PCR or sequencing
• Comet assay for DNA damage
• Oil red O staining

Correct Answer: RISC immunoprecipitation (RIP) followed by RT‑PCR or sequencing

Q15. Which clinical property of RISC activity explains why some siRNA therapeutics have prolonged pharmacodynamic effects despite rapid plasma clearance?

• RISC is rapidly degraded after each catalytic cycle
• RISC‑bound guide strand acts catalytically and is long‑lived within cells
• siRNA is converted to DNA and integrates into the genome
• siRNA causes permanent methylation of target gene promoters

Correct Answer: RISC‑bound guide strand acts catalytically and is long‑lived within cells

Q16. Which of the following is a known clinical siRNA that uses GalNAc conjugation to lower serum PCSK9 and reduce LDL cholesterol?

• Patisiran (Onpattro)
• Inclisiran (Leqvio)
• Givosiran (Givlaari)
• Nusinersen (Spinraza)

Correct Answer: Inclisiran (Leqvio)

Q17. In siRNA sequence design to minimize off‑targeting, which strategy is most effective?

• Maximizing seed complementarity to many 3’UTRs
• Avoiding seed matches to human transcriptome and using chemical seed modifications
• Designing fully complementary sequences to multiple genes
• Using only unmodified RNA to enhance potency

Correct Answer: Avoiding seed matches to human transcriptome and using chemical seed modifications

Q18. Which preclinical technique provides transcriptome‑wide assessment of on‑ and off‑target effects after siRNA treatment?

• Northern blot for single transcripts only
• RNA‑seq (next‑generation sequencing of RNA)
• Immunohistochemistry for proteins
• Liquid chromatography of metabolites

Correct Answer: RNA‑seq (next‑generation sequencing of RNA)

Q19. Which modification to an siRNA duplex can reduce recognition by endosomal Toll‑like receptors and lower immunostimulatory potential?

• Adding CpG motifs in the sequence
• Incorporating 2′-O‑methyl modifications in the guide strand seed region
• Lengthening the duplex to >50 bp
• Removing 3′ overhangs entirely

Correct Answer: Incorporating 2′-O‑methyl modifications in the guide strand seed region

Q20. Which manufacturing method is primarily used to produce chemically modified siRNAs at scale for clinical use?

• Recombinant expression in E. coli followed by protein extraction
• Chemical solid‑phase oligonucleotide synthesis with automated cycles
• Viral vector production and packaging
• In vitro transcription with T7 polymerase without modifications

Correct Answer: Chemical solid‑phase oligonucleotide synthesis with automated cycles

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