Systematic Evolution of Ligands by Exponential Enrichment
Encyclopedia
SELEX also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry
technique in molecular biology
for producing oligonucleotide
s of either single-stranded DNA
or RNA
that specifically bind to a target ligand
or ligands.. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB (selected and amplified binding site) and CASTing (Cyclic amplification and selection of targets)
The process begins with the synthesis of a very large oligonucleotide library consisting of randomly generated sequences of fixed length flanked by constant 5' and 3' ends that serve as primers
. For a randomly generated region of length n, the number of possible sequences in the library is 4n.(Four nucleotides (A,T,C,G), with n possibilities). The sequences in the library are exposed to the target ligand - which may be a protein
or a small organic compound - and those that do not bind the target are removed, usually by affinity chromatography
. The bound sequences are eluted and amplified by PCR
to prepare for subsequent rounds of selection in which the stringency of the elution conditions is increased to identify the tightest-binding sequences. An advancement on the original method allows an RNA library to omit the constant primer regions, which can be difficult to remove after the selection process because they stabilize secondary structure
s that are unstable when formed by the random region alone.
The technique has been used to evolve aptamers of extremely high binding affinity to a variety of target ligands, including small molecule
s such as ATP
and adenosine and proteins such as prion
s and vascular endothelial growth factor
(VEGF). Clinical uses of the technique are suggested by aptamers that bind tumor marker
s and a VEGF-binding aptamer trade-named Macugen has been approved by the FDA for treatment of macular degeneration
.
One caution advanced in relation to the method emphasizes that selection for extremely high, sub-nano
molar binding affinities may not in fact improve specificity for the target molecule. Off-target binding to related molecules could have significant clinical effects.
Combinatorial chemistry
Combinatorial chemistry involves the rapid synthesis or the computer simulation of a large number of different but structurally related molecules or materials...
technique in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
for producing oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s of either single-stranded DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
that specifically bind to a target ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
or ligands.. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB (selected and amplified binding site) and CASTing (Cyclic amplification and selection of targets)
The process begins with the synthesis of a very large oligonucleotide library consisting of randomly generated sequences of fixed length flanked by constant 5' and 3' ends that serve as primers
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
. For a randomly generated region of length n, the number of possible sequences in the library is 4n.(Four nucleotides (A,T,C,G), with n possibilities). The sequences in the library are exposed to the target ligand - which may be a protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
or a small organic compound - and those that do not bind the target are removed, usually by affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
. The bound sequences are eluted and amplified by PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
to prepare for subsequent rounds of selection in which the stringency of the elution conditions is increased to identify the tightest-binding sequences. An advancement on the original method allows an RNA library to omit the constant primer regions, which can be difficult to remove after the selection process because they stabilize secondary structure
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
s that are unstable when formed by the random region alone.
The technique has been used to evolve aptamers of extremely high binding affinity to a variety of target ligands, including small molecule
Small molecule
In the fields of pharmacology and biochemistry, a small molecule is a low molecular weight organic compound which is by definition not a polymer...
s such as ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
and adenosine and proteins such as prion
Prion
A prion is an infectious agent composed of protein in a misfolded form. This is in contrast to all other known infectious agents which must contain nucleic acids . The word prion, coined in 1982 by Stanley B. Prusiner, is a portmanteau derived from the words protein and infection...
s and vascular endothelial growth factor
Vascular endothelial growth factor
Vascular endothelial growth factor is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate....
(VEGF). Clinical uses of the technique are suggested by aptamers that bind tumor marker
Tumor marker
A tumor marker is a substance found in the blood, urine, or body tissues that can be elevated in cancer, among other tissue types. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer...
s and a VEGF-binding aptamer trade-named Macugen has been approved by the FDA for treatment of macular degeneration
Macular degeneration
Age-related macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field because of damage to the retina. It occurs in “dry” and “wet” forms. It is a major cause of blindness and visual impairment in older adults...
.
One caution advanced in relation to the method emphasizes that selection for extremely high, sub-nano
Nano
Nano- is a prefix meaning a billionth. Used primarily in the metric system, this prefix denotes a factor of 10−9 or . It is frequently encountered in science and electronics for prefixing units of time and length, such as 30 nanoseconds , 100 nanometres or in the case of electrical capacitance,...
molar binding affinities may not in fact improve specificity for the target molecule. Off-target binding to related molecules could have significant clinical effects.