A gapmer is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage.
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.