Types Aptamer

1 types

1.1 nucleic acid
1.2 peptides

1.2.1 affimer

types
nucleic acid

nucleic acid aptamers nucleic acid species have been engineered through repeated rounds of in vitro selection or equivalently, selex (systematic evolution of ligands exponential enrichment) bind various molecular targets such small molecules, proteins, nucleic acids, , cells, tissues , organisms. aptamers useful in biotechnological , therapeutic applications offer molecular recognition properties rival of commonly used biomolecule, antibodies. in addition discriminate recognition, aptamers offer advantages on antibodies can engineered in test tube, readily produced chemical synthesis, possess desirable storage properties, , elicit little or no immunogenicity in therapeutic applications.

in 1990, 2 labs independently developed technique of selection: gold lab, using term selex process of selecting rna ligands against t4 dna polymerase; , szostak lab, coining term in vitro selection, selecting rna ligands against various organic dyes. szostak lab coined term aptamer (from latin, apto, meaning fit ) these nucleic acid-based ligands. 2 years later, szostak lab , gilead sciences, independent of 1 another, used in vitro selection schemes evolve single stranded dna ligands organic dyes , human coagulant, thrombin (see anti-thrombin aptamers), respectively. there not appear systematic differences between rna , dna aptamers, save greater intrinsic chemical stability of dna.

interestingly enough, notion of selection in vitro preceded twenty-plus years prior when sol spiegelman used qbeta replication system way evolve self-replicating molecule. in addition, year before publishing of in vitro selection , selex, gerald joyce used system termed directed evolution alter cleavage activity of ribozyme.

since discovery of aptamers, many researchers have used aptamer selection means application , discovery. in 2001, process of in vitro selection automated j. colin cox in ellington lab @ university of texas @ austin, reducing duration of selection experiment 6 weeks 3 days.

while process of artificial engineering of nucleic acid ligands highly interesting biology , biotechnology, notion of aptamers in natural world had yet uncovered until 2002 when 2 groups led ronald breaker , evgeny nudler discovered nucleic acid-based genetic regulatory element (which named riboswitch) possesses similar molecular recognition properties artificially made aptamers. in addition discovery of new mode of genetic regulation, adds further credence notion of rna world , postulated stage in time in origins of life on earth.

both dna , rna aptamers show robust binding affinities various targets. dna , rna aptamers have been selected same target. these targets include lysozyme, thrombin, human immunodeficiency virus trans-acting responsive element (hiv tar), hemin, interferon γ, vascular endothelial growth factor (vegf), prostate specific antigen (psa), dopamine, , non-classical oncogene, heat shock factor 1 (hsf1). in case of lysozyme, hiv tar, vegf , dopamine dna aptamer analog of rna aptamer, thymine replacing uracil. hemin, thrombin, , interferon γ, dna , rna aptamers selected through independent selections , have unique sequences. considering not dna analogs of rna aptamers show functionality, correlation between dna , rna sequence , structure , function requires further investigation.

lately, concept of smart aptamers, , smart ligands in general, has been introduced. describes aptamers selected pre-defined equilibrium (




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) constants , thermodynamic (Δh, Δs) parameters of aptamer-target interaction. kinetic capillary electrophoresis technology used selection of smart aptamers. obtains aptamers in few rounds of selection.

recent developments in aptamer-based therapeutics have been rewarded in form of first aptamer-based drug approved u.s. food , drug administration (fda) in treatment age-related macular degeneration (amd), called macugen offered osi pharmaceuticals. in addition, company neoventures biotechnology inc. has commercialized first aptamer based diagnostic platform analysis of mycotoxins in grain. many contract companies develop aptamers , aptabodies replace antibodies in research, diagnostic platforms, drug discovery, , therapeutics.

non-modified aptamers cleared rapidly bloodstream, half-life of minutes hours, due nuclease degradation , clearance body kidneys, result of aptamer s inherently low molecular weight. unmodified aptamer applications focus on treating transient conditions such blood clotting, or treating organs such eye local delivery possible. rapid clearance can advantage in applications such in vivo diagnostic imaging. example tenascin-binding aptamer under development schering ag cancer imaging. several modifications, such 2 -fluorine-substituted pyrimidines, polyethylene glycol (peg) linkage, etc. (both of used in macugen, fda-approved aptamer) available scientists increase serum half-life of aptamers day or week time scale.

another approach increase nuclease resistance of aptamers develop spiegelmers, composed entirely of unnatural l-ribonucleic acid backbone. spiegelmer of same sequence has same binding properties of corresponding rna aptamer, except binds mirror image of target molecule.

in addition development of aptamer-based therapeutics, many researchers such ellington lab have been developing diagnostic techniques aptamer based plasma protein profiling called aptamer plasma proteomics. technology enable future multi-biomarker protein measurements can aid diagnostic distinction of disease versus healthy states.

furthermore, hirao lab applied genetic alphabet expansion using unnatural base pair selex , achieved generation of high affinity dna aptamers. few hydrophobic unnatural base fifth base augment aptamer affinity target proteins.

as resource in vitro selection , selex experiments, ellington lab has developed aptamer database cataloging published experiments.

peptides

peptide aptamers artificial proteins selected or engineered bind specific target molecules. these proteins consist of 1 or more peptide loops of variable sequence displayed protein scaffold. typically isolated combinatorial libraries , subsequently improved directed mutation or rounds of variable region mutagenesis , selection. in vivo, peptide aptamers can bind cellular protein targets , exert biological effects, including interference normal protein interactions of targeted molecules other proteins. libraries of peptide aptamers have been used mutagens , in studies in investigator introduces library expresses different peptide aptamers cell population, selects desired phenotype, , identifies aptamers cause phenotype. investigator uses aptamers baits, example in yeast two-hybrid screens identify cellular proteins targeted aptamers. such experiments identify particular proteins bound aptamers, , protein interactions aptamers disrupt, cause phenotype. in addition, peptide aptamers derivatized appropriate functional moieties can cause specific postranslational modification of target proteins, or change subcellular localization of targets.

peptide aptamers can recognize targets in vitro. have found use in lieu of antibodies in biosensors , used detect active isoforms of proteins populations containing both inactive , active protein forms. derivatives known tadpoles, in peptide aptamer heads covalently linked unique sequence double-stranded dna tails , allow quantification of scarce target molecules in mixtures pcr (using, example, quantitative real-time polymerase chain reaction) of dna tails.

the peptides form aptamer variable regions synthesized part of same polypeptide chain scaffold , constrained @ n , c termini linkage it. double structural constraint decreases diversity of conformations variable regions can adopt, , reduction in conformational diversity lowers entropic cost of molecular binding when interaction target causes variable regions adopt single conformation. consequence, peptide aptamers can bind targets tightly, binding affinities comparable shown antibodies (nanomolar range).

peptide aptamer scaffolds typically small, ordered, soluble proteins. first scaffold, still used, escherichia coli thioredoxin, trxa gene product (trxa). in these molecules, single peptide of variable sequence displayed instead of gly-pro motif in trxa -cys-gly-pro-cys- active site loop. improvements trxa include substitution of serines flanking cysteines, prevents possible formation of disulfide bond @ base of loop, introduction of d26a substitution reduce oligomerization, , optimization of codons expression in human cells,. reviews in 2015 have reported studies using 12 , 20 other scaffolds.

peptide aptamer selection can made using different systems, used yeast two-hybrid system. peptide aptamers can selected combinatorial peptide libraries constructed phage display , other surface display technologies such mrna display, ribosome display, bacterial display , yeast display. these experimental procedures known biopannings. among peptides obtained biopannings, mimotopes can considered kind of peptide aptamers. peptides panned combinatorial peptide libraries have been stored in special database name mimodb.

selection of ligand regulated peptide aptamers (lirpas) has been demonstrated. displaying 7 amino acid peptides novel scaffold protein based on trimeric fkbp-rapamycin-frb structure, interaction between randomized peptide , target molecule can controlled small molecule rapamycin or non-immunosuppressive analogs.

affimer

the affimer protein, evolution of peptide aptamers, small, highly stable protein engineered display peptide loops provides high affinity binding surface specific target protein. protein of low molecular weight, 12–14 kda, derived cysteine protease inhibitor family of cystatins.

the affimer scaffold stable protein based on cystatin protein fold. displays 2 peptide loops , n-terminal sequence can randomised bind different target proteins high affinity , specificity similar antibodies. stabilisation of peptide upon protein scaffold constrains possible conformations peptide may take, increasing binding affinity , specificity compared libraries of free peptides.

the affimer protein scaffold developed @ mrc cancer cell unit in cambridge across 2 laboratories @ university of leeds. affimer technology has been commercialised , developed avacta life sciences, developing reagents research , therapeutic applications.