“…Length of the random region of the starting library is normally between 20 to 40 bp [11]. Modified nucleotides could be also included in the library, which may greatly broaden the range of possible sequences and probably enhance their in vivo stability or nuclease resistance [24]. The design of the conserved primer regions is also important as improper design would introduce unspecific products in PCR.…”
Section: Cell-selex Strategy and Proceduresmentioning
SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.
“…Length of the random region of the starting library is normally between 20 to 40 bp [11]. Modified nucleotides could be also included in the library, which may greatly broaden the range of possible sequences and probably enhance their in vivo stability or nuclease resistance [24]. The design of the conserved primer regions is also important as improper design would introduce unspecific products in PCR.…”
Section: Cell-selex Strategy and Proceduresmentioning
SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.
“…2'F, 2'OMe, and 2'NH 2 nucleotides have been incorporated into RNA aptamers with Y639F mutant T7 RNA polymerase [1]. Other chemically modified nucleotides such as 2'-deoxy-2'-fluoroarabinonucleotide (FANA) [11], 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA)[12], and C2'-O-methyl(C2'-OMe)/ C2'-Fluorine (C2'-F) [13] have been incorporated into aptamers with engineered polymerase. The 2'-O-carbamoyl uridine (U cm ) is successfully incorporated by a wild-type T7 RNA polymerase [14].…”
“…Therminator DNA polymerase was used to extend a DNA hairpin primer/template with the modified nucleotides, permitting amplification of the DNA primer-template after selection and resynthesis of the selected TNA aptamer. Kuwahara and co-workers used a mixture of mutant and/or wild type KOD DNA polymerases to “transcribe” and “reverse transcribe” mixed LNA/DNA oligonucleotides during selection of an anti-thrombin aptamer [15,16]. Holliger and co-workers have dramatically expanded the potential of these analogs by evolving a series of TgoT DNA polymerase mutants to better “transcribe” and “reverse transcribe” these modified nucleotides, and they have used them to select HNA aptamers targeting HIV trans-activating response RNA and hen egg lysozyme [17•], a FANA aptamer targeting HIV-1 RT [18], and catalytic nucleic acids containing ANA, FANA, HNA, and CeNA moieties [19].…”
Section: Selex With Modified Rna and Dnamentioning
DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.
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