View source for Biology:Systematic evolution of ligands by exponential enrichment

{{Short description|Technique for producing oligonucleotides that specifically bind to a target}}
[[File:SELEX schematic.png|375x375 px|right|thumb|A schematic of the major phases in a SELEX experiment. This cycle, may be repeated up to 20 times over a period lasting weeks, though some methods require far fewer cycles.]]
[[File:Aptamer biotin.png|thumb |Structure of an RNA aptamer specific for [[Chemistry:Biotin|biotin]]. The aptamer surface and backbone are shown in yellow. Biotin (spheres) fits snugly into a cavity of the RNA surface.|375x375 px]]
'''Systematic evolution of ligands by exponential enrichment''' ('''SELEX'''), also referred to as ''[[Biology:Deoxyribozyme#in vitro selection|in vitro selection]]'' or ''[[Biology:Deoxyribozyme#in vitro evolution|in vitro evolution]]'', is a [[Chemistry:Combinatorial chemistry|combinatorial chemistry]] technique in [[Biology:Molecular biology|molecular biology]] for producing [[Chemistry:Oligonucleotide|oligonucleotide]]s of either single-stranded [[Biology:DNA|DNA]] or [[Biology:RNA|RNA]] that specifically bind to a target [[Biology:Ligand (biochemistry)|ligand]] or ligands. These single-stranded [[Biology:DNA|DNA]] or [[Biology:RNA|RNA]] are commonly referred to as [[Biology:Aptamer|aptamer]]s.<ref>{{cite journal | vauthors = Hak-Hagir A | title = [The Hak-Hagir skin conduit] | journal = Zeitschrift für Urologie und Nephrologie | year = 1978 | volume = 71 | issue = 9 | pages = 639–642 | pmid= 362762 }}</ref><ref name=":0">{{cite journal | vauthors = Tuerk C, Gold L | title = Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase | journal = Science | volume = 249 | issue = 4968 | pages = 505–10 | date = August 1990 | pmid = 2200121 | doi = 10.1126/science.2200121 | bibcode = 1990Sci...249..505T }}</ref><ref name=":1">{{cite journal | vauthors = Ellington AD, Szostak JW | title = In vitro selection of RNA molecules that bind specific ligands | journal = Nature | volume = 346 | issue = 6287 | pages = 818–22 | date = August 1990 | pmid = 1697402 | doi = 10.1038/346818a0 | bibcode = 1990Natur.346..818E | s2cid = 4273647 }}</ref> 
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)<ref>{{cite journal | vauthors = Blackwell TK, Weintraub H | s2cid = 1995608 | title = Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection | journal = Science | volume = 250 | issue = 4984 | pages = 1104–10 | date = November 1990 | pmid = 2174572 | doi = 10.1126/science.2174572 | bibcode = 1990Sci...250.1104B }}</ref><ref>{{cite journal | vauthors = Wright WE, Binder M, Funk W | title = Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site | journal = Molecular and Cellular Biology | volume = 11 | issue = 8 | pages = 4104–10 | date = August 1991 | pmid = 1649388 | pmc = 361222 | doi = 10.1128/mcb.11.8.4104 }}</ref> SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.<ref>{{cite journal | vauthors = Gold L | title = SELEX: How It Happened and Where It will Go | journal = Journal of Molecular Evolution | volume = 81 | issue = 5–6 | pages = 140–143 | date = December 2015 | pmid = 26480964 | pmc = 4661202 | doi = 10.1007/s00239-015-9705-9 | bibcode = 2015JMolE..81..140G }}</ref>

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. The constant ends serve as [[Biology:Primer (molecular biology)|primers]], while a small number of random regions are expected to bind specifically to the chosen target. For a randomly generated region of length ''n'', the number of possible sequences in the library using conventional DNA or RNA is 4<sup>n</sup> (''n'' positions with four possibilities (A,T,C,G) at each position). The sequences in the library are exposed to the target ligand - which may be a [[Biology:Protein|protein]] or a small organic compound - and those that do not bind the target are removed, usually by [[Physics:Affinity chromatography|affinity chromatography]] or target capture on paramagnetic beads.<ref name=":4">{{cite journal | vauthors = Stoltenburg R, Schubert T, Strehlitz B | title = In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A | journal = PLOS ONE | volume = 10 | issue = 7 | pages = e0134403 | date = 2015-07-29 | pmid = 26221730 | pmc = 4519192 | doi = 10.1371/journal.pone.0134403 | bibcode = 2015PLoSO..1034403S | doi-access = free }}</ref> The bound sequences are eluted and amplified by [[Biology:Polymerase chain reaction|PCR]]<ref name=":0" /><ref name=":1" /> to prepare for subsequent rounds of selection in which the stringency of the elution conditions can be increased to identify the tightest-binding sequences.<ref name=":0" /> A caution to consider in this method is that the selection of extremely high, sub-[[Nano-|nano]]molar binding affinity entities may not in fact improve specificity for the target molecule.<ref name="Carothers">{{cite journal | vauthors = Carothers JM, Oestreich SC, Szostak JW | title = Aptamers selected for higher-affinity binding are not more specific for the target ligand | journal = Journal of the American Chemical Society | volume = 128 | issue = 24 | pages = 7929–37 | date = June 2006 | pmid = 16771507 | pmc = 4287982 | doi = 10.1021/ja060952q }}</ref> Off-target binding to related molecules could have significant clinical effects.

SELEX has been used to develop a number of aptamers that bind targets interesting for both clinical and research purposes.<ref name=":3">{{cite journal | vauthors = Wu YX, Kwon YJ | title = Aptamers: The "evolution" of SELEX | journal = Methods | volume = 106 | pages = 21–8 | date = August 2016 | pmid = 27109056 | doi = 10.1016/j.ymeth.2016.04.020 }}</ref> Nucleotides with chemically modified sugars and bases have been incorporated into SELEX reactions to increase the chemical diversity at each base, expanding the possibilities for specific and sensitive binding, or increasing stability in [[Biology:Serum (blood)|serum]] or ''[[Biology:In vivo|in vivo]]''.<ref name=":3" /><ref name=":2">{{cite journal | vauthors = Keefe AD, Cload ST | title = SELEX with modified nucleotides | journal = Current Opinion in Chemical Biology | volume = 12 | issue = 4 | pages = 448–56 | date = August 2008 | pmid = 18644461 | doi = 10.1016/j.cbpa.2008.06.028 }}</ref>

== Procedure ==
Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use.<ref>{{cite journal | vauthors = Shorie M, Kaur H | title = Microtitre Plate Based Cell-SELEX Method | journal = Bio-Protocol | volume = 8 | issue = 20 | pages = e3051 | date = October 2018 | pmid = 34532522 | pmc = 8342047 | doi = 10.21769/BioProtoc.3051 }}</ref>

=== Generating single stranded oligonucleotide library ===
The first step of SELEX involves the synthesis of fully or partially randomized oligonucleotide sequences of some length flanked by defined regions which allow PCR amplification of those randomized regions and, in the case of RNA SELEX, in vitro transcription of the randomized sequence.<ref name=":0" /><ref name=":1" /><ref name="Huizenga2">{{cite journal | vauthors = Huizenga DE, Szostak JW | title = A DNA aptamer that binds adenosine and ATP | journal = Biochemistry | volume = 34 | issue = 2 | pages = 656–65 | date = January 1995 | pmid = 7819261 | doi = 10.1021/bi00002a033 }}</ref> While Ellington and Szostak demonstrated that chemical synthesis is capable of generating ~10<sup>15</sup> unique sequences for oligonucleotide libraries in their 1990 paper on in vitro selection,<ref name=":1" /> they found that amplification of these synthesized oligonucleotides led to significant loss of pool diversity due to PCR bias and defects in synthesized fragments.<ref name=":1" /> The oligonucleotide pool is amplified and a sufficient amount of the initial library is added to the reaction so that there are numerous copies of each individual sequence to minimize the loss of potential binding sequences due to stochastic events.<ref name=":1" /> Before the library is introduced to target for incubation and selective retention, the sequence library must be converted to single stranded oligonucleotides to achieve structural conformations with target binding properties.<ref name=":0" /><ref name=":1" />

=== Target incubation ===
Immediately prior to target introduction, the single stranded oligonucleotide library is often heated and cooled slowly to renature oligonucleotides into thermodynamically stable secondary and tertiary structures.<ref name=":1" /><ref name=":4" /> Once prepared, the randomized library is incubated with immobilized target to allow oligonucleotide-target binding. There are several considerations for this target incubation step, including the target immobilization method and strategies for subsequent unbound oligonucleotide separation, incubation time and temperature, incubation buffer conditions, and target versus oligonucleotide concentrations. Examples of target immobilization methods include [[Physics:Affinity chromatography|affinity chromatography]] columns,<ref name=":1" /> nitrocellulose [[Biology:Filter binding assay|binding assay filters]],<ref name=":0" /> and [[Physics:Dynabeads|paramagnetic beads.]]<ref name=":4" /> Recently, SELEX reactions have been developed where the target is whole cells, which are expanded near complete [[Biology:Confluency|confluence]] and incubated with the oligonucleotide library on culture plates.<ref name=":5">{{cite journal | vauthors = Iwagawa T, Ohuchi SP, Watanabe S, Nakamura Y | title = Selection of RNA aptamers against mouse embryonic stem cells | journal = Biochimie | volume = 94 | issue = 1 | pages = 250–7 | date = January 2012 | pmid = 22085640 | doi = 10.1016/j.biochi.2011.10.017 }}</ref> Incubation buffer conditions are altered based on the intended target and desired function of the selected aptamer. For example, in the case of negatively charged small molecules and proteins, high salt buffers are used for [[Physics:Electric-field screening|charge screening]] to allow nucleotides to approach the target and increase the chance of a specific binding event.<ref name=":1" /> Alternatively, if the desired aptamer function is in vivo protein or whole cell binding for potential therapeutic or diagnostic application, incubation buffer conditions similar to in vivo plasma salt concentrations and homeostatic temperatures are more likely to generate aptamers that can bind in vivo. Another consideration in incubation buffer conditions is non-specific competitors. If there is a high likelihood of non-specific oligonucleotide retention in the reaction conditions, non specific competitors, which are small molecules or polymers other than the SELEX library that have similar physical properties to the library oligonucleotides, can be used to occupy these non-specific binding sites.<ref name=":5" /> Varying the relative concentration of target and oligonucleotides can also affect properties of the selected aptamers. If a good binding affinity for the selected aptamer is not a concern, then an excess of target can be used to increase the probability that at least some sequences will bind during incubation and be retained. However, this provides no selective pressure for high [[Biology:Ligand (biochemistry)|binding affinity]], which requires the oligonucleotide library to be in excess so that there is competition between unique sequences for available specific binding sites.<ref name=":0" />

=== Binding sequence elution and amplification ===
Once the oligonucleotide library has been incubated with target for sufficient time, unbound oligonucleotides are washed away from immobilized target, often using the incubation buffer so that specifically bound oligonucleotides are retained.<ref name=":1" /> With unbound sequences washed away, the specifically bound sequences are then eluted by creating denaturing conditions that promote oligonucleotide unfolding or loss of binding conformation including flowing in deionized water,<ref name=":1" /> using denaturing solutions containing urea and EDTA,<ref name=":5" /><ref name=":6">{{cite journal | vauthors = Vater A, Jarosch F, Buchner K, Klussmann S | title = Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX | journal = Nucleic Acids Research | volume = 31 | issue = 21 | pages = 130e–130 | date = November 2003 | pmid = 14576330 | pmc = 275487 | doi = 10.1093/nar/gng130 }}</ref> or by applying high heat and physical force.<ref name=":4" /> Upon elution of bound sequences, the retained oligonucleotides are [[Biology:Reverse transcriptase|reverse-transcribed]] to DNA in the case of RNA or modified base selections,<ref name=":0" /><ref name=":1" /><ref name=":5" /> or simply collected for amplification in the case of DNA SELEX.<ref name=":7">{{cite journal | vauthors = Blank M, Weinschenk T, Priemer M, Schluesener H | title = Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen | journal = The Journal of Biological Chemistry | volume = 276 | issue = 19 | pages = 16464–8 | date = May 2001 | pmid = 11279054 | doi = 10.1074/jbc.M100347200 | s2cid = 39002909 | doi-access = free }}</ref> These DNA templates from eluted sequences are then amplified via [[Biology:Polymerase chain reaction|PCR]] and converted to single stranded DNA, RNA, or modified base oligonucleotides, which are used as the initial input for the next round of selection.<ref name=":0" /><ref name=":1" />

===Obtaining ssDNA===
One of the most critical steps in the SELEX procedure is obtaining single stranded DNA (ssDNA) after the PCR amplification step. This will serve as input for the next cycle so it is of vital importance that all the DNA is single stranded and as little as possible is lost. Because of the relative simplicity, one of the most used methods is using biotinylated reverse primers in the amplification step, after which the complementary strands can be bound to a resin followed by elution of the other strand with lye. Another method is asymmetric PCR, where the amplification step is performed with an excess of forward primer and very little reverse primer, which leads to the production of more of the desired strand. A drawback of this method is that the product should be purified from double stranded DNA (dsDNA) and other left-over material from the PCR reaction. Enzymatic degradation of the unwanted strand can be performed by tagging this strand using a phosphate-probed primer, as it is recognized by enzymes such as [[Biology:Exodeoxyribonuclease|Lambda exonuclease]]. These enzymes then selectively degrade the phosphate tagged strand leaving the complementary strand intact. All of these methods recover approximately 50 to 70% of the DNA. For a detailed comparison refer to the article by Svobodová et al. where these, and other, methods are experimentally compared.<ref name="Svobodová">{{cite journal | vauthors = Svobodová M, Pinto A, Nadal P, O' Sullivan CK | title = Comparison of different methods for generation of single-stranded DNA for SELEX processes | journal = Analytical and Bioanalytical Chemistry | volume = 404 | issue = 3 | pages = 835–42 | date = August 2012 | pmid = 22733247 | doi = 10.1007/s00216-012-6183-4 | s2cid = 206910212 }}</ref> In classical SELEX, the process of randomized single stranded library generation, target incubation, and binding sequence elution and amplification described above are repeated until the vast majority of the retained pool consists of target binding sequences,<ref name=":0" /><ref name=":1" /> though there are modifications and additions to the procedure that are often used, which are discussed below.

=== Negative or counter selection ===
In order to increase the specificity of aptamers selected by a given SELEX procedure, a negative selection, or counter selection, step can be added prior to or immediately following target incubation. To eliminate sequences with affinity for target immobilization matrix components from the pool, negative selection can be used where the library is incubated with target immobilization matrix components and unbound sequences are retained.<ref name=":6" /><ref>{{cite journal | vauthors = Stoltenburg R, Reinemann C, Strehlitz B | title = SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands | journal = Biomolecular Engineering | volume = 24 | issue = 4 | pages = 381–403 | date = October 2007 | pmid = 17627883 | doi = 10.1016/j.bioeng.2007.06.001 }}</ref><ref name=":7" /> Negative selection can also be used to eliminate sequences that bind target-like molecules or cells by incubating the oligonucleotide library with small molecule target analogs, undesired cell types, or non-target proteins and retaining the unbound sequences.<ref name=":5" /><ref name=":7" /><ref>{{cite journal | vauthors = Haller AA, Sarnow P | title = In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 94 | issue = 16 | pages = 8521–6 | date = August 1997 | pmid = 9238009 | pmc = 22984 | doi = 10.1073/pnas.94.16.8521 | bibcode = 1997PNAS...94.8521H | doi-access = free }}</ref>

=== Tracking selection progression ===
To track the progress of a SELEX reaction, the number of target bound molecules, which is equivalent to the number of oligonucleotides eluted, can be compared to the estimated total input of oligonucleotides following elution at each round.<ref name=":1" /><ref name=":8">{{cite journal | vauthors = Sefah K, Shangguan D, Xiong X, O'Donoghue MB, Tan W | title = Development of DNA aptamers using Cell-SELEX | language = En | journal = Nature Protocols | volume = 5 | issue = 6 | pages = 1169–85 | date = June 2010 | pmid = 20539292 | doi = 10.1038/nprot.2010.66 | s2cid = 4953042 }}</ref> The number of eluted oligonucleotides can be estimated through elution concentration estimations via 260&nbsp;nm wavelength absorbance<ref name=":8" /> or fluorescent labeling of oligonucleotides.<ref name=":4" /> As the SELEX reaction approaches completion, the fraction of the oligonucleotide library that binds target approaches 100%, such that the number of eluted molecules approaches the total oligonucleotide input estimate, but may converge at a lower number.<ref name=":1" />

=== Caveats and considerations ===
Some SELEX reactions can generate probes that are dependent on primer binding regions for secondary structure formation.<ref name=":4" /> There are aptamer applications for which a short sequence, and thus primer truncation, is desirable.<ref name=":9">{{cite journal | vauthors = Lubin AA, Hunt BV, White RJ, Plaxco KW | title = Effects of probe length, probe geometry, and redox-tag placement on the performance of the electrochemical E-DNA sensor | language = EN | journal = Analytical Chemistry | volume = 81 | issue = 6 | pages = 2150–8 | date = March 2009 | pmid = 19215066 | doi = 10.1021/ac802317k }}</ref> 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 [[Biology:Secondary structure|secondary structure]]s that are unstable when formed by the random region alone.<ref name="Jarosch2">{{cite journal | vauthors = Jarosch F, Buchner K, Klussmann S | title = In vitro selection using a dual RNA library that allows primerless selection | journal = Nucleic Acids Research | volume = 34 | issue = 12 | pages = e86 | date = July 2006 | pmid = 16855281 | pmc = 1524915 | doi = 10.1093/nar/gkl463 }}</ref>

== Chemically modified nucleotides ==
Recently, SELEX has expanded to include the use of chemically modified nucleotides. These chemically modified oligonucleotides offer many potential advantages for selected aptamers including greater stability and nuclease resistance, enhanced binding for select targets, expanded physical properties - like increased hydrophobicity, and more diverse structural conformations.<ref name=":3" /><ref name=":2" /><ref>{{cite journal | vauthors = Pinheiro VB, Taylor AI, Cozens C, Abramov M, Renders M, Zhang S, Chaput JC, Wengel J, Peak-Chew SY, McLaughlin SH, Herdewijn P, Holliger P | title = Synthetic genetic polymers capable of heredity and evolution | journal = Science | volume = 336 | issue = 6079 | pages = 341–4 | date = April 2012 | pmid = 22517858 | pmc = 3362463 | doi = 10.1126/science.1217622 | bibcode = 2012Sci...336..341P }}</ref>

The genetic alphabet, and thus possible aptamers, is also expanded using unnatural base pairs<ref>{{cite journal | vauthors = Kimoto M, Kawai R, Mitsui T, Yokoyama S, Hirao I | title = An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules | journal = Nucleic Acids Research | volume = 37 | issue = 2 | pages = e14 | date = February 2009 | pmid = 19073696 | pmc = 2632903 | doi = 10.1093/nar/gkn956 }}</ref><ref>{{cite journal | vauthors = Yamashige R, Kimoto M, Takezawa Y, Sato A, Mitsui T, Yokoyama S, Hirao I | title = Highly specific unnatural base pair systems as a third base pair for PCR amplification | journal = Nucleic Acids Research | volume = 40 | issue = 6 | pages = 2793–806 | date = March 2012 | pmid = 22121213 | pmc = 3315302 | doi = 10.1093/nar/gkr1068 }}</ref> the use of these unnatural base pairs was applied to SELEX and high affinity DNA aptamers were generated.<ref>{{cite journal | vauthors = Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I | title = Generation of high-affinity DNA aptamers using an expanded genetic alphabet | journal = Nature Biotechnology | volume = 31 | issue = 5 | pages = 453–7 | date = May 2013 | pmid = 23563318 | doi = 10.1038/nbt.2556 | s2cid = 23329867 }}</ref>

== SELEX variants and alternative aptamer selection methods ==
FRELEX was developed in 2016 by [https://neoaptamers.com/ NeoVentures Biotechnology Inc] to allow the selection of aptamers without immobilizing the target or the oligonucleotide library.<ref name=":12">{{Cite patent|number=10415034|title=United States Patent: 10415034 - Method for the selection of aptamers for unbound targets|gdate=September 17, 2019|invent1=Penner|invent2=CA|inventor1-first=Gregory|url=https://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=/netahtml/PTO/searchbool.html&r=3&f=G&l=50&co1=AND&d=PTXT&s1=aptamer&s2=penner.INNM.&OS=aptamer+AND+IN/penner&RS=aptamer+AND+IN/penner}}</ref> Immobilization is a necessary component of SELEX; however, it has the potential to inhibit key epitopes, and thus weaken the likelihood of successful binding, particularly when working with small molecules.<ref>{{cite journal |vauthors=Kohlberger M, Gadermaier G |date=August 2021 |title=SELEX: Critical factors and optimization strategies for successful aptamer selection |journal=Biotechnology and Applied Biochemistry |volume=69 |issue=5 |pages=1771–1792 |doi=10.1002/bab.2244 |pmid=34427974|pmc=9788027 |s2cid=237280042 }}</ref><ref>{{cite journal |vauthors=Klapak D, Broadfoot S, Penner G, Singh A, Inapuri E |date=2018-10-11 |title=Development of novel aptamers for low-density lipoprotein particle quantification |journal=PLOS ONE |volume=13 |issue=10 |pages=e0205460 |bibcode=2018PLoSO..1305460K |doi=10.1371/journal.pone.0205460 |pmc=6181373 |pmid=30307996 |doi-access=free}}</ref> FRELEX follows a similar overall methodology to SELEX; however, instead of immobilizing the target, the researcher introduces a series of random and blocker oligonucleotides to an immobilization field before introduction to the target.<ref name=":12" /> This allows the researcher to better target small molecules that may be lost during partitioning.<ref name=":12" /> It also can be used in some circumstances to select an aptamer library without knowing the target.<ref>{{cite journal |vauthors=Lecocq S, Spinella K, Dubois B, Lista S, Hampel H, Penner G |date=2018-01-05 |title=Aptamers as biomarkers for neurological disorders. Proof of concept in transgenic mice |journal=PLOS ONE |volume=13 |issue=1 |pages=e0190212 |bibcode=2018PLoSO..1390212L |doi=10.1371/journal.pone.0190212 |pmc=5755763 |pmid=29304088 |doi-access=free |veditors=de Franciscis V}}</ref>

== Prior targets ==
The technique has been used to evolve [[Biology:Aptamer|aptamer]]s of extremely high binding affinity to a variety of target ligands, including [[Biology:Small molecule|small molecule]]s such as [[Chemistry:Adenosine triphosphate|ATP]]<ref name="Dieckmann">{{cite journal | vauthors = Dieckmann T, Suzuki E, Nakamura GK, Feigon J | title = Solution structure of an ATP-binding RNA aptamer reveals a novel fold | journal = RNA | volume = 2 | issue = 7 | pages = 628–40 | date = July 1996 | pmid = 8756406 | pmc = 1369402 }}</ref> and [[Chemistry:Adenosine|adenosine]]<ref name="Huizenga2" /><ref name="Burke">{{cite journal | vauthors = Burke DH, Gold L | title = RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX | journal = Nucleic Acids Research | volume = 25 | issue = 10 | pages = 2020–4 | date = May 1997 | pmid = 9115371 | pmc = 146680 | doi = 10.1093/nar/25.10.2020 }}</ref> and proteins such as [[Medicine:Prion|prion]]s<ref name="Mercey">{{cite journal | vauthors = Mercey R, Lantier I, Maurel MC, Grosclaude J, Lantier F, Marc D | title = Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns | journal = Archives of Virology | volume = 151 | issue = 11 | pages = 2197–214 | date = November 2006 | pmid = 16799875 | doi = 10.1007/s00705-006-0790-3 | s2cid = 32195593 }}</ref> and [[Biology:Vascular endothelial growth factor|vascular endothelial growth factor]] (VEGF).<ref name="Ulrich">{{cite journal | vauthors = Ulrich H, Trujillo CA, Nery AA, Alves JM, Majumder P, Resende RR, Martins AH | title = DNA and RNA aptamers: from tools for basic research towards therapeutic applications | journal = Combinatorial Chemistry & High Throughput Screening | volume = 9 | issue = 8 | pages = 619–32 | date = September 2006 | pmid = 17017882 | doi = 10.2174/138620706778249695 }}</ref> Moreover, SELEX has been used to select high-affinity aptamers for complex targets such as tumor cells,<ref>{{cite journal | vauthors = Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L | title = A tenascin-C aptamer identified by tumor cell SELEX: systematic evolution of ligands by exponential enrichment | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 26 | pages = 15416–21 | date = December 2003 | pmid = 14676325 | pmc = 307582 | doi = 10.1073/pnas.2136683100 | bibcode = 2003PNAS..10015416D | doi-access = free }}</ref><ref>{{cite journal | vauthors = Mayer G, Ahmed MS, Dolf A, Endl E, Knolle PA, Famulok M | title = Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures | journal = Nature Protocols | volume = 5 | issue = 12 | pages = 1993–2004 | date = December 2010 | pmid = 21127492 | doi = 10.1038/nprot.2010.163 | s2cid = 4984082 }}</ref> tumor exosomes,<ref>{{cite journal | vauthors = Domenyuk V, Zhong Z, Stark A, Xiao N, O'Neill HA, Wei X, Wang J, Tinder TT, Tonapi S, Duncan J, Hornung T, Hunter A, Miglarese MR, Schorr J, Halbert DD, Quackenbush J, Poste G, Berry DA, Mayer G, Famulok M, Spetzler D | display-authors = 6 | title = Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach | journal = Scientific Reports | volume = 7 | issue = 1 | pages = 42741 | date = February 2017 | pmid = 28218293 | pmc = 5316983 | doi = 10.1038/srep42741 | bibcode = 2017NatSR...742741D }}</ref><ref>{{cite journal | vauthors = Hornung T, O'Neill HA, Logie SC, Fowler KM, Duncan JE, Rosenow M, Bondre AS, Tinder T, Maher V, Zarkovic J, Zhong Z, Richards MN, Wei X, Miglarese MR, Mayer G, Famulok M, Spetzler D | display-authors = 6 | title = ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes | journal = Nucleic Acids Research | volume = 48 | issue = 8 | pages = 4013–4027 | date = May 2020 | pmid = 31989173 | pmc = 7192620 | doi = 10.1093/nar/gkaa034 }}</ref> or tumor tissue.<ref>{{cite journal | vauthors = Domenyuk V, Gatalica Z, Santhanam R, Wei X, Stark A, Kennedy P, Toussaint B, Levenberg S, Wang J, Xiao N, Greil R, Rinnerthaler G, Gampenrieder SP, Heimberger AB, Berry DA, Barker A, Quackenbush J, Marshall JL, Poste G, Vacirca JL, Vidal GA, Schwartzberg LS, Halbert DD, Voss A, Magee D, Miglarese MR, Famulok M, Mayer G, Spetzler D | display-authors = 6 | title = Poly-ligand profiling differentiates trastuzumab-treated breast cancer patients according to their outcomes | journal = Nature Communications | volume = 9 | issue = 1 | pages = 1219 | date = March 2018 | pmid = 29572535 | pmc = 5865185 | doi = 10.1038/s41467-018-03631-z | bibcode = 2018NatCo...9.1219D }}</ref> Clinical uses of the technique are suggested by aptamers that bind tumor markers,<ref name="Ferreira">{{cite journal | vauthors = Ferreira CS, Matthews CS, Missailidis S | title = DNA aptamers that bind to MUC1 tumour marker: design and characterization of MUC1-binding single-stranded DNA aptamers | journal = Tumour Biology | volume = 27 | issue = 6 | pages = 289–301 | year = 2006 | pmid = 17033199 | doi = 10.1159/000096085 | s2cid = 41664944 }}</ref> GFP-related fluorophores,<ref name="PaigeWu2011">{{cite journal | vauthors = Paige JS, Wu KY, Jaffrey SR | title = RNA mimics of green fluorescent protein | journal = Science | volume = 333 | issue = 6042 | pages = 642–6 | date = July 2011 | pmid = 21798953 | pmc = 3314379 | doi = 10.1126/science.1207339 | bibcode = 2011Sci...333..642P }}</ref> and a VEGF-binding aptamer trade-named [[Chemistry:Pegaptanib|Macugen]] has been approved by the FDA for treatment of [[Medicine:Macular degeneration|macular degeneration]].<ref name="Ulrich" /><ref name="Vavvas">{{cite journal | vauthors = Vavvas D, D'Amico DJ | title = Pegaptanib (Macugen): treating neovascular age-related macular degeneration and current role in clinical practice | journal = Ophthalmology Clinics of North America | volume = 19 | issue = 3 | pages = 353–60 | date = September 2006 | pmid = 16935210 | doi = 10.1016/j.ohc.2006.05.008 | doi-broken-date = 1 August 2023 }}</ref> Additionally, SELEX has been utilized to obtain highly specific catalytic DNA or DNAzymes. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific),<ref>{{cite journal | vauthors = Breaker RR, Joyce GF | title = A DNA enzyme that cleaves RNA | journal = Chemistry & Biology | volume = 1 | issue = 4 | pages = 223–9 | date = December 1994 | pmid = 9383394 | doi = 10.1016/1074-5521(94)90014-0 }}</ref> the CA1-3 DNAzymes (copper-specific),<ref>{{cite journal | vauthors = Carmi N, Shultz LA, Breaker RR | title = In vitro selection of self-cleaving DNAs | journal = Chemistry & Biology | volume = 3 | issue = 12 | pages = 1039–46 | date = December 1996 | pmid = 9000012 | doi = 10.1016/s1074-5521(96)90170-2 | doi-access = free }}</ref> the 39E DNAzyme (uranyl-specific) <ref>{{cite journal | vauthors = Liu J, Brown AK, Meng X, Cropek DM, Istok JD, Watson DB, Lu Y | title = A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 7 | pages = 2056–61 | date = February 2007 | pmid = 17284609 | pmc = 1892917 | doi = 10.1073/pnas.0607875104 | bibcode = 2007PNAS..104.2056L | doi-access = free }}</ref> and the NaA43 DNAzyme (sodium-specific).<ref>{{cite journal | vauthors = Torabi SF, Wu P, McGhee CE, Chen L, Hwang K, Zheng N, Cheng J, Lu Y | title = In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 112 | issue = 19 | pages = 5903–8 | date = May 2015 | pmid = 25918425 | pmc = 4434688 | doi = 10.1073/pnas.1420361112 | bibcode = 2015PNAS..112.5903T | doi-access = free }}</ref>

These developed aptamers have seen diverse application in therapies for macular degeneration<ref name="Vavvas" /> and various research applications including [[Biology:Biosensor|biosensor]]s,<ref name=":9" /> fluorescent labeling of proteins<ref>{{Cite journal | vauthors = Umrao S, Jain V, Chakraborty B, Roy R | title = Protein-induced fluorescence enhancement as aptamer sensing mechanism for thrombin detection. | journal = Sensors and Actuators B: Chemical | date = August 2018 | volume = 267 | pages = 294–301 | doi = 10.1016/j.snb.2018.04.039 | s2cid = 103202899 }}</ref> and cells,<ref>{{cite journal | vauthors = Terazono H, Anzai Y, Soloviev M, Yasuda K | title = Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases | journal = Journal of Nanobiotechnology | volume = 8 | issue = 1 | pages = 8 | date = April 2010 | pmid = 20388214 | pmc = 2861636 | doi = 10.1186/1477-3155-8-8 | doi-access = free }}</ref> and selective enzyme inhibition.<ref>{{cite journal | vauthors = Mondragón E, Maher LJ | title = RNA aptamer inhibitors of a restriction endonuclease | journal = Nucleic Acids Research | volume = 43 | issue = 15 | pages = 7544–55 | date = September 2015 | pmid = 26184872 | pmc = 4551934 | doi = 10.1093/nar/gkv702 }}</ref>

== See also ==
* {{annotated link|Biology:Aptamer}}
* {{annotated link|Biology:Deoxyribozyme}}
* {{annotated link|Chemistry:Anti-thrombin aptamers}}
* {{annotated link|Biology:Bacterial one-hybrid system}}

== References ==
{{reflist}}

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Levine HA, Nilsen-Hamilton M | title = A mathematical analysis of SELEX | journal = Computational Biology and Chemistry | volume = 31 | issue = 1 | pages = 11–35 | date = February 2007 | pmid = 17218151 | pmc = 2374838 | doi = 10.1016/j.compbiolchem.2006.10.002 }}
* {{cite journal | vauthors = Spill F, Weinstein ZB, Irani Shemirani A, Ho N, Desai D, Zaman MH | title = Controlling uncertainty in aptamer selection | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 43 | pages = 12076–12081 | date = October 2016 | pmid = 27790993 | pmc = 5087011 | doi = 10.1073/pnas.1605086113 | arxiv = 1612.08995 | bibcode = 2016PNAS..11312076S | doi-access = free }}
{{refend}}

== External links ==
* [http://aptamer.freebase.com Aptamer Base]


[[Category:Evolution]]
[[Category:Molecular biology]]

{{Sourceattribution|Systematic evolution of ligands by exponential enrichment}}