Try out PMC Labs and tell us what you think. Learn More. Escort aptamers are DNA or RNA sequences with high affinity to certain cell-surface proteins, which can be used for targeted delivery of various agents into cells of a definite type. The peculiarities of the selection of escort aptamers are discussed in this review. The methods used in selection of escort aptamers via the SELEX technique are considered, including selection against isolated cell-surface proteins, cell fragments, living eukaryotic cells, and bacteria.
Particular attention is given to the de and chemical modification of escort aptamers. The different fields of application of escort aptamers are described, including the targeted delivery of siRNAs, nanoparticles, toxins, and photoagents, as well as the identification of specific cell markers and the detection or isolation of cells of a definite type.
The potential for the application of escort aptamers in the development of new therapeutic agents and diagnostic systems is also discussed. Aptamers Latin aptus — suitable are single-stranded DNA and RNA molecules that are capable of specific recognition of definite types of compounds, thanks to their unique spatial structure.
In the s, methods for in vitro selection, enabling one to obtain nucleic acids with predetermined properties, were described by three independent research groups. Ellington and J. Szostak [ 1 ] obtained an RNA molecule that was capable of specifically binding to an organic dye. Tuerk and L. Robertson and G. Joyce used in vitro selection to convert a group I ribozyme from a ribonuclease into a deoxyribonuclease [ 3 ]. Throughout the subsequent two decades, this field has developed rapidly; methods for the selection of aptamers and approaches to their de have been further refined.
A large of aptamers capable of binding to various targets with high specificity have already been obtained see Reviews [4— 7 ]. Aptamers find broad application across a wide range of research fields, thanks to their unique properties namely their high affinity and selectivity in binding to a target molecule.
In particular, aptamers can be used to obtain highly efficient and specific inhibitors of target proteins that can be applied in the de of new drugs. A of aptamers are currently in different stages of clinical trials [ 8 ].
Macugen Eyetech Pharmaceuticals and Pfizerwhich is based on aptamer binding a human vascular endothelial growth factor VEGFhas been certified as an efficient drug for the treatment of age-related macular degeneration [9, 10 ]. One of the most interesting and promising aspects in the field is deing aptamers that are capable of specific recognition of cells of a definite type through binding with certain dominants on their surface.
In the review by B. Hicke et al. The use of escort aptamers as an addressing fragment opens wide possibilities for the targeted delivery of agents of different nature to cells of definite types.
Today, a large of escort aptamers directed toward various target cells have been obtained, and a wide range of applications for these aptamers for specific action on cells, diagnostics, and cell isolation have been described. The present review is devoted to the selection, de, and different aspects in the use of escort aptamers. DNA and RNA aptamers are obtained via in vitro selection from combinatorial libraries of nucleic acid molecules.
A conventional library is a set of oligonucleotides with the randomized region consisting of 20—60 nucleotides flanked with the constant regions that are required for binding to primers and the PCR amplification of DNA. RNA aptamers are capable of forming a greater variety of spatial structures as compared with DNA aptamers, as a result of the presence of 2'-OH groups.
However, RNA aptamers are more sensitive to the action of cell nucleases and require the introduction of additional protective groups [ 12 ]. The ssDNA libraries are obtained via the conventional methods for the chemical synthesis of oligodeoxyribonucleotides using a mixture of all four monomers when synthesizing a randomized fragment. The general scheme of the SELEX method for DNA libraries comprises the following stages: incubation of a library with a target, separation of oligonucleotide—target complexes from the unbound oligonucleotides, disruption of the oligonucleotide—target complexes, and amplification of the bound molecules Fig.
During the selection process, the library is enriched in sequences possessing increased affinity to the target. Five to fifteen rounds of selection are typically performed to obtain aptamers, depending on the values of the dissociation constant of the aptamer—target complex.
After the dissociation constant ceases to decrease i. The homology between the individual aptamers is then analyzed. On the basis of the obtained, the aptamers are classified into several groups; their capability to interact with the target is assessed. The sequences with the maximum affinity to the target are selected for further studies.
The secondary structure of aptamers is studied by analyzing their conserved motifs, by computer simulation, and chemical and enzymatic probing. The minimal size of an aptamer required for specific recognition of a target is determined at the next stage.
For this purpose, a series of truncated variants of the aptamer is synthesized and the ability of these aptamers to bind to the target is determined. Aptamers are usually characterized by high affinity to their targets. In terms of their affinity and specificity, aptamers are similar to monoclonal antibodies; however, aptamers have a of distinct characterisitcs Table 1.
Among these characteristics, the possibilities to produce an aptamer via chemical synthesis and to modify them chemically are the ones most worthy of note. The introduction of different modifications into escort aptamers enables one to considerably increase their stability in biological media, as well as their functionality. The introduction of different substituents at the 2' position of ribose Fig. This type of modification is typically used to protect escort RNA aptamers, whereas escort DNA aptamers are more frequently used without any additional modifications. The major pathway of degradation of RNA aptamers in biological media is cleavage by pyrimidine endoribonucleases; therefore, as early as at the stage of construction of combinatorial RNA libraries for selecting escort aptamers, the pyrimidine nucleosides within them are substituted for their 2'-fluoro- and 2'-amino analogues by using the corresponding modified nucleoside triphosphates for synthesizing a library.
It is possible to use T7 RNA polymerase [ 13 ] or its mutant version capable of inserting these 2'-modified nucleoside triphosphates into RNA with higher efficiency to integrate them into a growing RNA strand [ 14 ].
There is also mutant RNA polymerase inserting 2'-O-methyl analogues of nucleoside triphosphates [15, 16 ]; however, due to the problems associated with the reaction of reverse transcription of 2'-O-methyl-containing RNAs, the use of 2'-O-methyl-RNA libraries directly during the selection process has not yet become common practice [ 4 ].
Chemical modifications of sugar-phosphate backbone increasing the resistance of escort aptamers in biological media.
In order to obtain 2'-O-methyl-containing aptamers, a quantity of ribonucleotides within a RNA aptamer are substituted for their 2'-O-methyl analogues, after the aptamers have been selected and their nucleotide sequences determined. The introduction of 2'-O,4'-C-methylene-linked bicyclic nucleotides LNA — locked nucleic acids is another way of increasing the stability of aptamers of a known sequence.
Capping of the 3'-terminus with an additional thymidine residue linked via the 3'-3'-phosphodiester bond is used to prevent the cleavage of aptamers [17, 19 ]. In the case of escort aptamers, the introduction of an aliphatic amino or sulfhydryl group to the 5'- or 3'-terminus of an aptamer is the most common.
It allows one to synthesize the various conjugates of aptamers with toxins, antibiotics, fluorescent or photoreactive groups, nanoparticles, etc. Therefore, in each case one must thoroughly select the type and position of these modifications. When selecting escort aptamers in vitrothe individual proteins from the cell surface or whole cells are used as targets.
The use of cells as targets has a of advantages over using purified proteins:.
The protocol for cell selection of escort aptamers has specific features. The high of surface dominants, which can either be unique for a definite cell type or be common to cells of various types, is one of the key problems that arise when using cells as targets.
In order to eliminate the nonspecific aptamers binding to the molecular targets that are common to many cell types from the selection, an additional stage of counter-selection, or negative selection, is added to SELEX. Sequential incubation of the nucleic acid library with the control cells and target cells enables one to select particular sequences that bind only to the desired protein on the cell surface.
The general scheme of cell selection of escort aptamers by the example of an RNA library is provided in Fig. The initial oligonucleotide library is incubated with control cells, and the unbound molecules are isolated.
They are then incubated with the target cells, and the unbound molecules are isolated again. After the cells have been disintegrated, the bound aptamers are extracted, amplified, and subsequently used in the next round of selection.
The scheme of in vitro cell selection including the negative selection stage by the example of the RNA library. The usage of cells as targets for in vitro selection was first described by K. Morris et al. DNA aptamers recognizing ghosts of human erythrocytes haemoglobin-free cells that retain the same shape of the membrane as native erythrocytes were obtained in that study.
In order to produce aptamers, the ssDNA library was incubated with target cells. Then, the bound sequences were isolated via filtration through nitrocellulose filters.
The resulting set of DNA molecules was amplified and used in the subsequent round of selection. Photoactivatable phenyl azide groups introduced into these two aptamers were used to demonstrate that they bind to the cell surface of various molecular targets.
The study was the first example of using combinatorial libraries of nucleic acids for the selection of aptamers targeted at such complex objects as the cell membrane. The overwhelming majority of studies devoted to the selection of aptamers targeting living cells have focused on the search for sequences that can specifically bind to malignant cells. To this end, S. Lupold et al. The cells of healthy tissues are characterized by a very low level of PSMA, which considerably increases with the development of malignant tumors.
A recombinant protein corresponding to the extracellular domain of PSMA, rather than whole tumor cells, was used as a target. A nt aptamer A was obtained via minimization of the length of the A10 aptamer; an additional thymidine residue was linked to the 3'-terminus of A via a 3'-3'-phosphodiester bond for the purpose of protection from exonucleases.
Ferreira et al. Mucin hyperexpression is typical of cancer cells. Immunogenic synthetic peptides mucin fragments immobilized on a column with functionalized sepharose were used as targets for the selection. After 10 rounds of selection, 12 aptameric sequences were obtained, one of which aptamer S1. ZIP: 66030