siRNA and microRNA: roles and importance MCQs With Answer

Introduction:
This quiz set on siRNA and microRNA: roles and importance is designed for M.Pharm students to deepen understanding of RNA interference pathways, molecular mechanisms, and therapeutic applications. Questions explore biogenesis, processing enzymes (Drosha, Dicer, Argonaute), target recognition, and distinctions between siRNA and miRNA. Clinical aspects include delivery strategies, chemical modifications for stability and specificity, immune responses, and approved siRNA drugs. Expect questions that test conceptual knowledge and practical considerations for RNA-based therapeutics, off-target effects, and experimental design. Use this quiz to evaluate readiness for advanced coursework, research projects, or pharmacological development involving small RNA technologies.

Q1. Which enzyme is primarily responsible for cropping primary microRNA (pri-miRNA) into precursor miRNA (pre-miRNA) in the nucleus?

• Exportin-5
• Dicer
• Drosha
• Argonaute 2

Correct Answer: Drosha

Q2. What is the main mechanistic difference between siRNA-mediated silencing and canonical miRNA-mediated regulation in animals?

• siRNA induces translational activation; miRNA causes mRNA methylation
• siRNA usually guides endonucleolytic mRNA cleavage via near-perfect complementarity; miRNA commonly causes translational repression or deadenylation with partial complementarity
• siRNA functions only in the nucleus; miRNA only in mitochondria
• siRNA requires Exportin-5 for function; miRNA does not

Correct Answer: siRNA usually guides endonucleolytic mRNA cleavage via near-perfect complementarity; miRNA commonly causes translational repression or deadenylation with partial complementarity

Q3. Which protein is the catalytic component of RISC that mediates target RNA cleavage in humans?

• Dicer
• Drosha
• Exportin-5
• Argonaute 2 (Ago2)

Correct Answer: Argonaute 2 (Ago2)

Q4. During siRNA duplex incorporation into RISC, which strand is typically retained as the guide strand?

• The strand with less stable 5′ end base-pairing (thermodynamically less stable at its 5′ end)
• The strand with greater GC content throughout
• The strand carrying a 5′ phosphate at higher concentration
• The longer strand if duplexes are asymmetric

Correct Answer: The strand with less stable 5′ end base-pairing (thermodynamically less stable at its 5′ end)

Q5. Exportin-5 is essential for which step in miRNA biogenesis?

• Cleavage of pri-miRNA into pre-miRNA
• Transport of pre-miRNA from nucleus to cytoplasm
• Loading of miRNA into Ago proteins
• Degradation of mature miRNA

Correct Answer: Transport of pre-miRNA from nucleus to cytoplasm

Q6. Which chemical modification is commonly used to increase siRNA stability and reduce immune stimulation in therapeutic applications?

• 5′ capping with m7G
• 2′-O-methylation of ribose
• Polyadenylation of the guide strand
• Thymidine to uridine substitution

Correct Answer: 2′-O-methylation of ribose

Q7. Patisiran and inclisiran are examples of clinically approved therapeutics that utilize which modality?

• Antisense oligonucleotides (ASOs)
• siRNA delivered via lipid nanoparticle or GalNAc conjugate
• CRISPR-Cas9 gene editing
• Monoclonal antibodies

Correct Answer: siRNA delivered via lipid nanoparticle or GalNAc conjugate

Q8. A major cause of off-target effects for siRNA in mammalian cells is:

• Activation of DNA repair pathways
• Seed-region complementarity of the guide strand to unintended 3′ UTRs resembling miRNA action
• Excessive methylation of the target gene promoter
• siRNA length being shorter than 19 nucleotides

Correct Answer: Seed-region complementarity of the guide strand to unintended 3′ UTRs resembling miRNA action

Q9. Which RNA sensing receptors are most commonly implicated in innate immune recognition of exogenous siRNA?

• Toll-like receptors 3, 7, and 8 (TLR3, TLR7, TLR8)
• NOD-like receptors only
• GPCR family receptors
• Receptor tyrosine kinases

Correct Answer: Toll-like receptors 3, 7, and 8 (TLR3, TLR7, TLR8)

Q10. Which structural feature best characterizes precursor miRNA (pre-miRNA)?

• Single-stranded linear RNA of ~200 nt with poly(A) tail
• Hairpin stem-loop of ~60–70 nt with a 2-nt 3′ overhang
• Circular RNA lacking free ends
• Double-stranded blunt-ended RNA duplex of 21 nt

Correct Answer: Hairpin stem-loop of ~60–70 nt with a 2-nt 3′ overhang

Q11. GW182 proteins function in miRNA-mediated repression primarily by:

• Cleaving target mRNA directly
• Recruiting deadenylation and decapping complexes to promote mRNA decay
• Exporting miRNA from the nucleus
• Methylating cytosines in miRNA seed sequences

Correct Answer: Recruiting deadenylation and decapping complexes to promote mRNA decay

Q12. In designing an siRNA for allele-specific silencing of a point mutation, which strategy increases selectivity against the mutant allele?

• Place the discriminatory nucleotide in the central region (positions 9–11) of the guide to exploit Ago2 cleavage sensitivity
• Ensure full complementarity to both wild-type and mutant alleles
• Prefer seed mismatch and central identity
• Use very short 15-nt siRNAs to reduce specificity

Correct Answer: Place the discriminatory nucleotide in the central region (positions 9–11) of the guide to exploit Ago2 cleavage sensitivity

Q13. Locked nucleic acids (LNAs) are introduced into small RNAs primarily to:

• Allow entry into mitochondria
• Increase binding affinity to target RNA and enhance nuclease resistance
• Trigger RNase H activity for cleavage of target RNA
• Promote nuclear retention of mature miRNAs

Correct Answer: Increase binding affinity to target RNA and enhance nuclease resistance

Q14. Which experimental observation best indicates that an siRNA acts through an Ago2-dependent slicer mechanism?

• Target mRNA levels remain unchanged while protein levels increase
• Generation of a cleavage product at the expected position 10–11 relative to the guide strand
• Complete nuclear retention of the target transcript
• Global upregulation of all miRNAs

Correct Answer: Generation of a cleavage product at the expected position 10–11 relative to the guide strand

Q15. Which delivery strategy is commonly used to target siRNA to hepatocytes in vivo?

• Cationic liposomes without targeting ligands
• GalNAc (N-acetylgalactosamine) conjugation for asialoglycoprotein receptor-mediated uptake
• Direct injection of naked siRNA into muscle only
• Use of hemagglutinin peptide to cross the blood–brain barrier

Correct Answer: GalNAc (N-acetylgalactosamine) conjugation for asialoglycoprotein receptor-mediated uptake

Q16. A hallmark distinction between plant and animal RNAi mechanisms is:

• Plants utilize Dicer, animals do not
• Plants often show systemic and transitive RNAi with RNA-dependent RNA polymerases (RdRPs); animals generally lack RdRP-dependent amplification
• Animals generate siRNAs but plants only use miRNAs
• Animal Ago proteins lack slicer activity entirely

Correct Answer: Plants often show systemic and transitive RNAi with RNA-dependent RNA polymerases (RdRPs); animals generally lack RdRP-dependent amplification

Q17. Which assay would best quantify mature miRNA levels in a tissue sample with high specificity?

• Northern blot using probes complementary to mature miRNA sequence
• Genomic DNA PCR
• Mass spectrometry of proteins
• Flow cytometry of whole cells without probes

Correct Answer: Northern blot using probes complementary to mature miRNA sequence

Q18. Why can high concentrations of exogenous siRNA saturate endogenous miRNA pathways in cells?

• Because siRNA increases cellular ATP levels
• Because RISC components (e.g., Ago proteins) and loading cofactors are limited, leading to competition between exogenous siRNA and endogenous miRNA for the same machinery
• Because siRNA converts into DNA that binds miRNA promoters
• Because siRNA directly degrades all miRNA genes

Correct Answer: Because RISC components (e.g., Ago proteins) and loading cofactors are limited, leading to competition between exogenous siRNA and endogenous miRNA for the same machinery

Q19. In small RNA sequencing data, a pronounced 5′ nucleotide bias (e.g., preference for uridine) of many miRNAs reflects:

• Sequencing machine error only
• Ago protein binding preferences and strand selection rules influencing stability and loading
• Random degradation patterns
• Exclusive expression of tRNA fragments instead of miRNAs

Correct Answer: Ago protein binding preferences and strand selection rules influencing stability and loading

Q20. For therapeutic siRNA targeting, which of the following design principles reduces the likelihood of unintended gene silencing?

• Design guide sequences with perfect matches to multiple transcripts
• Avoid seed region complementarity to abundant off-target 3′ UTR motifs and introduce chemical modifications in the seed to reduce miRNA-like off-targeting
• Use completely unmodified long double-stranded RNA (>200 nt)
• Favor GC-rich seed sequences to increase nonspecific binding

Correct Answer: Avoid seed region complementarity to abundant off-target 3′ UTR motifs and introduce chemical modifications in the seed to reduce miRNA-like off-targeting

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