Aptamers: problems, solutions and prospects

Abstract

Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally much less efficient than had been expected initially. This review is mostly devoted not to the successful use of aptamers but to the problems impeding the widespread application of aptamers in diagnostics and therapy, as well as to approaches that could considerably expand the range of aptamer application.

Keywords: SELEX; aptamer; diagnostics; problems; therapeutics.

Fig. 1

Scheme of SELEX. (a) IOP is incubated with a target molecule. (b) Unbound oligonucleotides are separated from bound molecules by washing steps. (c) Bound oligonucleotides are eluted from the target molecule. (d) Eluted oligonucleotides are amplified using the PCR (DNA-SELEX) or RT-PCR (RNA-SELEX) technique. (e) The enriched pool is then subjected to further rounds of selection. (f) After 5–15 rounds, aptamers are cloned and analyzed in detail

Fig. 2

Most frequently used modifications of nucleotides providing resistance of aptamers to nuclease degradation

Fig. 3

The structure of the first FDA-approved aptamer, Macugen. The following modified nucleotides were used: f – 2'-fluoronucleotide, m – 2'-O-methylnucleotide. The aptamer was conjugated to 40 kDa PEG to avoid quick excretion during renal filtration

Fig. 4

Aptamer displacement screening. This approach allows one to select small molecules competing with an aptamer for the same binding site

Fig. 5

Antidote-dependent regulation of aptamer functioning. The aptamer 9.3t is shown as an example [77]. This aptamer interacts with the coagulation factor IXa and has anticoagulation properties. Administration of a complementary antidote leads to quick inactivation of this aptamer and restoration of blood coagulation

Fig. 6

Scheme of Cell-SELEX. (a) IOP is first incubated with a nontarget cell in a negative selection step. (b) All oligonucleotides that show binding to the negative control cells are removed. (c) Unbound oligonucleotides from the negative step are added to the target cells in a positive selection step. (d) Unbound oligonucleotides from the positive step are separated from bound molecules by washing steps. (e) Oligonucleotides binding target cells are subsequently eluted. (f) Eluted oligonucleotides are amplified using the PCR (DNA-SELEX) or RT-PCR (RNA-SELEX) technique. (g) The enriched pool is then subjected to further rounds of selection. (h) After 15–20 rounds, aptamers are cloned and analyzed in detail