SELEX process is used for in vitro selection of aptamers from vast populations of random sequences. The previous methods of antisense technology and siRNA methods are limited in their applications. The functions such as target access, cell uptake and specificity of interaction are very limited in most of these methods rendering them unsuitable for wider applications.
Aptamers are oligonucleotide sequences which are assigned with specific functions. Aptamers are selected from random pools of DNA/RNA sequences based on their ability to bind. These sequences range typically from 30 to 100 nucleotides with specific binding sequences designed for binding with ligands. The new method of SELEX (systematic evolution of ligands by exponential enrichment) is a promising approach in this field. A variety of combinatorial techniques have been described thereafter.
The SELEX process was first developed by Turek and Gold in 1990. The term 'aptamer' was the contribution of two scientists Ellington and Szostak in the same year. The term has its origins in Greek word 'aptus' meaning 'to fit'. Aptamers find a variety of used in biotechnological, medicinal and other fields.
The SELEX method was developed simultaneously and independently by three laboratories in 1990. The method was primarily based on the properties such as degree of affinity of the synthetic oligonucleotide sequences to a given target, enzymatic activity etc. The process of selection and amplification was repeated till the pool of nucleotides largely consisted of the intended sequences which ensured directed evolution.
SELEX process essentially involves three major steps- Selection, Partitioning, and Amplification.
The experimental limit of the process is 1015 which refers to the number of oligonucleotide sequences which can be screened simultaneously. The process identifies a small group of sequences which can bind to the target. First the target molecule has to be identified and defined. It is followed by the process of selection of aptamer.
Selection of nucleotide sequences which bind specifically to the target site is the first and foremost step in the process. There are four factors which influence this step. Type of randomization used, chemistry of the nucleotide sequence, the length of the sequence and the purpose of the aptamer sequence.
The first step is the creation of a library of nucleotide sequences. Phosphoramidites in the ratio 1.5: 1.25: 1.15: 1.0 (A: C: G: U/T) is found to provide a balanced mix of nucleotide sequences. The length of the sequence determines the diversity of the nucleotide pool. With 'n' nucleotides, one can generate 4n different nucleotide sequences. The experimental limit of 1015 can identify oligonucleotide sequences of 25 nucleotides of length.
The nucleotide sequence pool is treated along with the target molecules/ligands with specific conditions of buffering and temperature. The ratio of nucleic acid: protein is kept as low as possible. This will ease the checking of the efficacy of binding to the target molecules.
This involves separating the aptamers from non aptamers. There are various techniques used in this step. Those nucleotide sequences which are not bound are separated. The RNA/DNA-aptamer complex is thus filtered out.
Partitioning with nitrocellulose is suited for all protein targets. Immobilization onto a solid matrix, immunoprecipitation etc are also employed. Immobilization technique can use different eluents which can improve the specificity of the aptamer.
For RNA selex, techniques such as nucleoside modifications using 2'-fluoro- or 2'-amino-CTP and -UTP rendering them immune to endonucleases or 5-iodo- or 5-bromo-UTP for crosslinking aptamers are useful.
This step is the final one aiming to amplify the aptamer sequences. This results in a distinct pool of targeted aptamer sequences. The usual PCR/RT-PCR technique and its modifications are used to amplify aptamers.
For RNA selex, techniques such as nucleoside modifications using 2'-fluoro- or 2'-amino-CTP and -UTP rendering them immune to endonucleases or 5-iodo- or 5-bromo-UTP for crosslinking aptamers are useful. These modifications make them more stable also. 2'-Fluoro-dCTP and 2'-FluorodUTP are also used in such modifications. For DNA SELEX, the pool of oligonucleotides is generated from the ssDNA strands of the PCR product. In vitro transcription is used to generate the random pool in RNA SELEX.
The entire cycle is repeated for higher precision. Each round of amplification give rise to variants which are different from the previous cycles and thus increasing the diversity of aptamers. These amplified molecules are used for the subsequent selection process. The initial selection process is usually stringent needing more incubation times. 6-20 cycles are usually required for proper selection of aptamers and then the selected sequences can be cloned into a vector and sequencing can be done.
The basic process of SELEX has seen many variations and combinations further improving the process of selection of aptamers in vitro.
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