11. Aptamers

What are aptamers

Aptamers are short, single-stranded DNA or RNA molecules that can bind to specific target molecules with high specificity. Aptamer comes from aptus ‘to fit’ and meros to ‘part’.

Aptamers target

proteins, peptides, small metal ion and organic molecules, viruses, bacteria, whole cells, targets within live animals

Secondary structure of aptamers

• Hairpin 

• Kissing complex

  

3D structure of aptamers

Combination of secondary structures form 3D structures capable of specific molecular recognition of target

Aptamers compared to RNA-based therapeutics

- Most therapeutic RNAs are designed to modulate RNA translation or (in the case of CRISPR), edit genes

- Aptamers assert their function by specific binding to a target molecule (often protein) and thereby blocking or activating (enhancing) molecular interactions

- Like antibodies (!?)

- Antibodies are dominating over aptamers

What does affinity and specificity of aptamers depend on?

3D shape and non-covalent interactions including hydrophobic and electrostatic, hydrogen bonding, van der Waal forces

DNA vs RNA aptamers

• Both RNA and ssDNA are capable of forming secondary structures 

• RNA aptamers form more diverse and intricate 3D structures, stronger intra-strand interactions (increased specificity and affinity), extra hydroxyl group 

• With RNA, smaller structures can be formed from the same number of nucleotides (size is important if used for penetrating tissues and cells), they can fold more easily 

• DNA aptamers are more stable (30-60 min half-life in serum vs a few seconds) because 

• RNA is a transient messenger-chemically unstable and degraded by nucleases 

• RNA aptamers are stabilized by chemical modification

• Selection process is more complex and expensive for RNA aptamers (advantage for DNA aptamers)

SELEX

It stands for Systemic Evolution of Ligands by Exponential enrichment. Gold-standard methodology for generating DNA or RNA aptamers

Advantages of aptamers as therapeutics

• Smaller and more flexible structure – can bind to smaller targets and hidden domains that might be inaccessible for antibodies, as well as toxic targets

• Can be raised against any target

• Cost-efficient and rapid in-vitro selection and production

• Controlled production with no batch effects and no animals involved

• Easily modifiable chemical structure 

• Low immunogenicity

Disadvantages of aptamers as therapeutics

• Short half-life

  • Solution: modifications, e.g., PEGylation or large, multivalent aptamers

• Limited toxicity

  • Potential toxicities include polyanionic effects, unexpected tissue accumulation and immunogenicity caused by chemical modifications/non-natural nucleotides

  • Polyanionic effect – highly negatively charged aptamers will bind to blood proteins → High uptake in non-target tissues

• Pharmacodynamic studies

Types of therapeutic aptamers

1. Antagonist – block the interaction of disease-associated targets

2. Agonist – activates the function of target receptors

3. Drug delivery system – carrier for other therapeutic agents

Aptamers vs antibodies

• Aptamers can reach inaccessible targets to antibodies due its lower molecular weight

• Many aptamers have been published and can be generated again without the need for SELEX → lowers cost for development/manufacturing

• Antibody has more batch-to-batch variation than aptamers

• Aptamers are more stable and have longer shelf life than antibodies 

• Aptamers have lower immunogenicity than antibodies. 

  • Targets for antibodies are limited to immunogenic molecules 

  • Targets for antibodies can’t be toxins or other molecules that do not cause a strong immune response

  • Antibodies partly circumvented using phage display

• Aptamers have faster kidney filtration than antibodies