A gapmer is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage.
Gapmers are chimeric RNA-DNA antisense oligonucleotide (ASO) hybrids containing a DNA core flanked by modified RNA residues (wings). The phosphate backbone is modified with phosphorothioate bonds to increase stability. Adding RNA modifications increases nuclease resistance and binding efficiency to complementary RNAs. The central DNA stretch serves as a guide for RNase H-mediated degradation of the complementary RNA.
The gapmer strategy allows the knockdown of targeted genes.
One of the key advantages of the gapmer strategy is the catalytic nature of RNase H degradation. This means that the ASO itself is recycled, enhancing the efficiency of the process. As a result, a single ASO can direct the degradation of multiple copies of the targeted RNA, a feat that a single steric blocking morpholino ASO (MO) cannot achieve as it can only bind and inactivate a single target RNA molecule (Pauli et al. 2015).
The general gapmer design consists of a 5’-wing followed by a gap of 8 to 12 deoxynucleic acid monomers that may contain a sulfur ion in the phosphor group (PS linkage) followed by a 3’-wing. A gapmer oligonucleotide is an RNA-DNA-RNA-like configuration. Inserting PS linkages improves the stability of the gapmer while maintaining its ability to elicit RNase H activity. Also, PS linkages contribute to protein binding properties that prevent rapid excretion and facilitate tissue uptake.
General Design
Designing gapmers involves several steps to ensure specificity and efficacy in binding to the target RNA. Gapmers are antisense oligonucleotides with a central DNA region flanked by modified RNA bases resistant to nuclease degradation that promote RNAse H-mediated cleavage of the target RNA. The modified nucleic acid bases 2’-OMe, 2’-MOE, and BNA(or LNA) are generally utilized in the design.
Steps to Design a Gapmer
Identify the Target RNA Sequence
Choose a specific RNA sequence as the target. This sequence could be a region within an mRNA or non-coding RNA. Select a sequence region accessible to hybridization. Regions to target are sequence stretches near the terminal end, internal loops, hairpins, and RNA bulges.
Select the Central DNA Region
The central DNA region, a pivotal element in the design of the gapmer, typically ranges from 8 to 12 nucleotides. This region is responsible for hybridizing with the target RNA and recruiting RNAse H for cleavage.
Design the Flanking Modified Bases
The flanking regions usually consist of 2-5 nucleotides on each side of the central DNA. These nucleotides are modified to enhance stability and binding affinity. Common modifications include 2'-O-methyl (2'-OMe) and bridged or locked nucleic acids (BNAs/LNAs).
Check for Specificity
It's crucial to ensure the gapmer sequence is specific to the target RNA, as this is a key step in minimizing off-target effects. This involves comparing the sequence against the transcriptome of the organism by performing a BLAST search.
Assess Secondary Structure
Analyze the secondary structure of the target RNA to ensure that the chosen binding site is accessible for hybridization.
Gapmer Designs Targeting PTEN
PTEN (Phosphatase and TENsin homolog): PTEN refers to the Phosphatase and TENsin homolog deleted on chromosome 10. PTEN is a tumor suppressor gene located in the 10q23 region of chromosome 10 encoding for a 403-amino acid multifunctional protein (predicted MW 47 kDa), which possesses lipid and protein phosphatase activities. PTEN can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate. However, whether PTEN is physiologically relevant to insulin signaling in vivo is unclear.
Prakash et al. (2010) reported that N-MeO-amino BNA, 2’,4’-BNANC, and 2’ ,4’-BNANC-[NMe] containing ASOs (14-mer 2-10-2 gapmer) showed high affinity to target RNA, significantly higher than the corresponding MOE ASO but similar to the LNA ASO.
The wings can be modified with MOE, LNA, and BNA moities. The backbone can be modified with phosphorothioates.
Published Design Examples
A O-MOE winged gapmer
5′-CTGCTAGCCTCTGGATTTGA-3′
A phosphorothioate chimeric oligonucleotide with 2′-O-methoxyethyl groups (wings) on bases 1–5 and 16–20.
Location of the target within cDNA (Butler et al. 2002).
>AK030750.1 Mus musculus 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730512F02 product:phosphatase and tensin homolog, full insert sequence
601 gatcttgaca aagcaaacaa agacaaggcc aaccgatact tctctccaaa ttttaaggtg
661 aaactatact ttacaaaaac agtagaggag ccatcaaatc cagaggctag cagttcaact
3’-AGTTTAG GTCTCCGATC GTC-5’
O-MOE PS Gapmer 5′-CTGCTAGCCTCTGGATTTGA-3′
721 tctgtgactc cagatgttag tgacaatgaa cctgatcatt atagatattc tgacaccact
781 gactctgatc cagagaatga accttttgat gaagatcagc attcacaaat tacaaaagtc
A BNA winged gapmer
d(CUTAGCACTGGCCU)3’; 2’,4’-BNANC[NMe] PTEN
A phosphorothioate chimeric oligonucleotide with bridged nucleic acid (BNA) groups (wings) on bases 1–2 and 13–14 (see also Gapmer-Design).
The PTEN gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The location of the antisense gapmer within the targeted sequence is shown for a mouse PTEN homolog mRNA.
Mus musculus phosphatase and tensin homolog (Pten), mRNA
NCBI Reference Sequence: NM_008960.2
2881 AAAGACACAG CAGCAATGAC TTAACCATAC AAATGTGGAG GCTTTCAACA AAGGATGGGC
2941 TGGAAACAGA AAATTTGACA ATGATTTATT CAGTATGCTT TCTCAGTTGT AATGACTGCT
3001 CCATCTCCTA TGTAATCAAG GCCAGTGCTA AGAGTCAGAT GCTATTAGTC CCTACATCAG
3’-TC CGGTCACGAT TC-5’10MER
5’-CT TAGCACTGGC CT-3’
BNA gapmer CU TAGCACTGGC CU
3061 TCAACACCTT ACCTTTATTT TTATTAATTT TCAATCATAT ACCTACTGTG GATGCTTCAT
3121 GTGCTGGCTG CCAGTTTGTT TTTCTCCTTA AATATTTTAT AATTCTTCAC AGGAAATTTC
Reference
BNA(Bridged-Nucleic-Acid-BNA3)-Design-Guidelines
BNA-gapmer-antisense-rna
BNA-PCSK9-gapmers
BNA-based-Oligonucleotide-Probes
Butler, M.; McKay, R. A.; Popoff, I. J.; Gaarde, W. A.; Witchell, D.; Murray, S. F.; Dean, N. M.; Bhanot, S.; Monia, B. P. Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice. Diabetes 2002, 51, 1028–1034.[Article]
Fluiter K, Mook OR, Vreijling J, Langkjaer N, Højland T, Wengel J, Baas F. Filling the gap in LNA antisense oligo gapmers: the effects of unlocked nucleic acid (UNA) and 4'-C-hydroxymethyl-DNA modifications on RNase H recruitment and efficacy of an LNA gapmer. Mol Biosyst. 2009 Aug;5(8):838-43. [Gapmer]
Gapmer-Design Example: PTEN ASOs.
PTEN Structure [pdb-1D5R]
Iacopetta D, Ceramella J, Baldino N, Sinicropi MS, Catalano A. Targeting Breast Cancer: An Overlook on Current Strategies. Int J Mol Sci. 2023 Feb 11;24(4):3643. [MDPI], [PMC]
Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, et al. (October 1999). "Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association". Cell. 99 (3): 323–334.
Liu BH, Jobichen C, Chia CSB, Chan THM, Tang JP, Chung TXY, Li J, Poulsen A, Hung AW, Koh-Stenta X, Tan YS, Verma CS, Tan HK, Wu CS, Li F, Hill J, Joy J, Yang H, Chai L, Sivaraman J, Tenen DG. Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide. Proc Natl Acad Sci U S A. 2018 Jul 24;115(30):E7119-E7128. [PMC]
Manning KS, Rao AN, Castro M, Cooper TA. BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low Toxicity in Myotonic Dystrophy Cells. ACS Chem Biol. 2017 Oct 20;12(10):2503-2509. [Pdf]
Milella M, Falcone I, Conciatori F, Cesta Incani U, Del Curatolo A, Inzerilli N, Nuzzo CM, Vaccaro V, Vari S, Cognetti F, Ciuffreda L. PTEN: Multiple Functions in Human Malignant Tumors. Front Oncol. 2015 Feb 16;5:24. [PMC]
Pauli A, Montague TG, Lennox KA, Behlke MA, Schier AF. Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish. PLoS One. 2015 Oct 5;10(10):e0139504. [PMC]
PTEN gene: [wiki-PTEN]
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