Antisense technologies, siRNA and antisense oligonucleotides MCQs With Answer

Introduction: Antisense technologies, including small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), are transformative approaches in modern drug discovery for M.Pharm students. These nucleic acid–based modalities selectively modulate gene expression by targeting specific RNA sequences to reduce, modify, or restore protein production. Understanding the principles behind sequence design, chemical modifications (e.g., phosphorothioate, 2’-O modifications, LNA, PMO), mechanisms (RNase H recruitment vs. RISC-mediated cleavage vs. steric blocking), delivery strategies (lipid nanoparticles, GalNAc conjugates), and safety issues (off-target effects, immune activation) is essential for therapeutic development. This MCQ set reinforces core concepts, design considerations, and clinical translation challenges relevant to antisense therapeutics.

Q1. Which mechanism is primarily responsible for degradation of target mRNA by gapmer antisense oligonucleotides?

• Recruitment of RISC complex leading to cleavage
• Steric blocking of ribosome assembly
• Activation of RNase H that cleaves RNA in RNA–DNA duplexes
• Induction of RNA editing through ADAR enzymes

Correct Answer: Activation of RNase H that cleaves RNA in RNA–DNA duplexes

Q2. Which chemical modification is commonly used to increase nuclease resistance and protein binding of ASOs while retaining RNase H activity in gapmers?

• Phosphorothioate backbone modification
• 2’-O-methyl full substitution on the entire strand
• Peptide nucleic acid (PNA) backbone
• Unlocked nucleic acid (UNA) throughout the oligo

Correct Answer: Phosphorothioate backbone modification

Q3. For siRNA therapeutics, what is the main role of Dicer in the RNA interference pathway?

• Direct binding to target mRNA to block translation
• Cleavage of long double-stranded RNA into ~21–23 nt siRNA duplexes
• Recruitment of RNase H to degrade RNA in RNA–DNA hybrids
• Conjugation of GalNAc to siRNA for hepatocyte delivery

Correct Answer: Cleavage of long double-stranded RNA into ~21–23 nt siRNA duplexes

Q4. Which delivery strategy is widely used clinically to deliver siRNA to hepatocytes by receptor-mediated uptake?

• Liposome complexation without targeting ligand
• GalNAc (N-acetylgalactosamine) conjugation targeting ASGPR
• Chitosan nanoparticles targeting integrins
• Direct intravenous injection of naked siRNA

Correct Answer: GalNAc (N-acetylgalactosamine) conjugation targeting ASGPR

Q5. Which antisense modality acts mainly by steric blockade of splicing or translation rather than by recruiting RNase H?

• Gapmers with central DNA region
• Steric-blocking ASOs such as morpholino oligomers (PMO)
• LNA–DNA antisense gapmers
• siRNA duplexes loaded into RISC

Correct Answer: Steric-blocking ASOs such as morpholino oligomers (PMO)

Q6. Which modification to the ribose sugar increases binding affinity to RNA and is often used in ASO wings to enhance potency?

• 2’-O-methyl (2’-OMe)
• Phosphodiester linkage
• Ribose hydroxylation at 3’ position
• Phosphorothioate substitution on every phosphate

Correct Answer: 2’-O-methyl (2’-OMe)

Q7. Which clinical siRNA therapeutic uses lipid nanoparticle (LNP) formulation for systemic delivery and targets transthyretin (TTR)?

• Nusinersen
• Patisiran
• Inclisiran
• Fomivirsen

Correct Answer: Patisiran

Q8. What is the primary safety concern related to phosphorothioate-modified ASOs at high doses?

• Complete lack of plasma protein binding leading to rapid clearance
• Sequence-independent activation of platelets and complement causing thrombocytopenia
• Universal immune tolerance with no adverse events
• Uncontrolled incorporation into genomic DNA causing mutagenesis

Correct Answer: Sequence-independent activation of platelets and complement causing thrombocytopenia

Q9. Which property of siRNA guide-strand selection is favored to ensure correct strand incorporation into RISC?

• Higher thermodynamic stability at the 5’ end of the guide strand
• Lower thermodynamic stability (weaker base-pairing) at the 5’ end of the guide strand
• Equal GC content throughout both strands
• Presence of a 5’ cap structure on the guide strand

Correct Answer: Lower thermodynamic stability (weaker base-pairing) at the 5’ end of the guide strand

Q10. Which innate immune receptors are most commonly implicated in recognizing single-stranded or double-stranded oligonucleotides and causing immune activation?

• Toll-like receptors (TLR7/8 for ssRNA and TLR3 for dsRNA)
• G-protein coupled receptors (GPCRs) on hepatocytes
• Nuclear hormone receptors like PPARγ
• Toll-like receptor 4 (TLR4) exclusively for DNA oligos

Correct Answer: Toll-like receptors (TLR7/8 for ssRNA and TLR3 for dsRNA)

Q11. What is the primary advantage of locked nucleic acid (LNA) modifications in ASO design?

• Complete elimination of protein binding to increase renal clearance
• Marked increase in thermal stability and binding affinity to complementary RNA
• Ability to recruit Dicer for siRNA production
• Conversion of ASOs into peptide-like molecules for cell penetration

Correct Answer: Marked increase in thermal stability and binding affinity to complementary RNA

Q12. Which assay is most appropriate to measure sequence-specific cleavage of target mRNA after ASO treatment in cultured cells?

• RNAse protection assay or qRT-PCR quantifying target mRNA levels
• ELISA for DNA repair enzymes
• Mass spectrometry of oligonucleotide mass only
• Colorimetric assay for glucose concentration

Correct Answer: RNAse protection assay or qRT-PCR quantifying target mRNA levels

Q13. Which ASO chemical class is charge-neutral and commonly used for steric-blocking applications such as exon skipping?

• Phosphorothioate-modified DNA
• Morpholino oligomers (PMO)
• 2’-O-methoxyethyl (2’-MOE) phosphodiester oligos
• Standard RNA oligonucleotides with phosphodiester backbone

Correct Answer: Morpholino oligomers (PMO)

Q14. What is a gapmer design in antisense oligonucleotides?

• A fully modified RNA oligo that blocks translation without RNase H
• An ASO with central DNA “gap” flanked by modified nucleotides to enable RNase H activity and protect ends
• A double-stranded siRNA mimic formed by two gap regions
• An ASO conjugated to GalNAc for hepatocyte uptake

Correct Answer: An ASO with central DNA “gap” flanked by modified nucleotides to enable RNase H activity and protect ends

Q15. Which clinical antisense drug is used intravitreally to treat cytomegalovirus retinitis and represents one of the earliest ASO therapies?

• Fomivirsen
• Nusinersen
• Patisiran
• Eteplirsen

Correct Answer: Fomivirsen

Q16. Which factor most strongly influences off-target effects of siRNA molecules?

• Length of the 3’ poly-A tail of the target mRNA
• Partial sequence complementarity (seed region matches) to unintended transcripts
• Total molecular weight of the siRNA duplex
• Presence of a 5’ phosphate on the passenger strand only

Correct Answer: Partial sequence complementarity (seed region matches) to unintended transcripts

Q17. Which pharmacokinetic property is typically prolonged for phosphorothioate ASOs compared with unmodified oligonucleotides?

• Rapid renal filtration and urine excretion within minutes
• Increased plasma protein binding leading to longer circulating half-life
• Immediate degradation by RNase H in plasma
• Complete inability to distribute into tissues

Correct Answer: Increased plasma protein binding leading to longer circulating half-life

Q18. What is the rationale for using asymmetric chemical modification of the two strands in siRNA duplexes?

• To ensure the passenger strand preferentially loads into RISC
• To promote guide-strand selection and reduce off-target effects by destabilizing the 5’ end of the guide or modifying the passenger strand
• To convert the duplex into a single-stranded ASO that recruits RNase H
• To make both strands equally active to double the on-target effect

Correct Answer: To promote guide-strand selection and reduce off-target effects by destabilizing the 5’ end of the guide or modifying the passenger strand

Q19. Which clinical ASO is approved for spinal muscular atrophy (SMA) and acts by modifying splicing of SMN2 pre-mRNA?

• Nusinersen
• Givosiran
• Patisiran
• Inclisiran

Correct Answer: Nusinersen

Q20. Which analytical method is essential during ASO/siRNA development to confirm correct sequence and detect degradation products?

• High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS)
• Gram staining
• Western blot for DNA-binding proteins only
• Flame photometry for sodium concentration

Correct Answer: High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS)

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