Methods for Isolation of Aptamers

In vitro selection,25 also called systematic evolution of ligands by exponential enrichment (SELEX),26'48 is a protocol to isolate rare functional oligonucleotides (i.e., aptamers) from a pool of random oligonucleotides (Figure 16.1). Similar to phage display or other strategies used to isolate ligands from random libraries, SELEX is essentially an iterative selection and amplification protocol to isolate single-stranded nucleic acid ligands that bind to their target with high affinity and specificity. The complexity of the starting library is determined in part by the number of random nucleotides in the pool. For example, by using a library with 40 random nucleotides, a pool of 1024 distinct nucleotides can be generated. Practically speaking, the number of ligands in the starting pool for in vitro selection is closer to 1015, representing 1 nmol of the library.

In the initial step, a library of random nucleotides flanked by fixed nucleotides is generated by solid phase oligonucleotide synthesis. The random nucleotides serve to add complexity to the pool while the fixed sequences are utilized for polymerase chain reaction (PCR) amplification. In the n° o _k n ^ —►

(1014-1015 oligonucleotides)

Nucleic acid library Fixed Random Fixed

Primer 1

ACGGACTACGGTTGAG-40N-CGAATGCGAACGTACAGT

Primer 2

(1014-1015 oligonucleotides)

ACGGACTACGGTTGAG-40N-CGAATGCGAACGTACAGT

Target molecule bound to a solid support

Isolation of bound nucleotides

Target molecule bound to a solid support

Repeat until maximum enrichment of ligands has been obtained

Reiterative rounds of selection and amplification

Cloning of oligonucleotides

Isolation of individual aptamers and characterization and amplification of plasmids and amplification of plasmids

FIGURE 16.1 Schematic representation of SELEX. An oligonucleotide library is synthesized containing random sequences that are flanked by fixed sequences that facilitate PCR amplification. Target molecules are incubated with this pool of oligonucleotides, and bound and unbound oligonucleotides are partitioned. Bound oligonucleotides are isolated, and iterative rounds of selection and amplification are performed with increased stringency to isolate aptamers with high specificity and affinity for the target molecule. Oligonucleo-tide ligands representing the aptamers are subsequently cloned in plasmids, amplified, and sequenced. The net result of this enrichment process is a small number of highly specific aptamers that are isolated from a large library of random oligonucleotides.

case of isolating DNA aptamers, the oligonucleotide pool is incubated with the target of interest, and the bound fragments are partitioned and directly amplified by PCR system. The resulting pool is used in a follow-up round of selection and amplification, and the process is repeated until the affinity for the target antigen plateaus. Typically, this will be achieved in six to ten rounds of SELEX. After the last round of SELEX, aptamers are cloned in plasmids, amplified, sequenced, and their binding constants are determined (Figure 16.1). These aptamers may be subject to additional modification such as size minimization to truncate the nucleotides not necessary for binding characteristics and nuclease stabilization by replacing naturally occurring nucleotides with modified nucleotides (i.e., 2'-F pyrimidines, 2'-OCH3 nucleotides) that are poor substrates for endo- and exonuclease degradation.

In contrast to the isolation of DNA aptamers, which require single-step amplification after portioning, the selection of RNA aptamers involves additional steps, including transcription of an RNA pool from the starting DNA library, reverse transcription of the partitioned RNA pool to generate a cDNA fragment and subsequent amplification of DNA, and transcription into RNA for the next round of selection.34 The advantage of RNA SELEX, however, is that unnatural nucleotides such as 2'-F pyrimidines and 2'-OCH3 nucleotides may be used in the transcription of the RNA pool because these modified bases are utilized by RNA polymerase as substrate. Furthermore, mutant RNA polymerases have also been described that are capable of improved incorporation of modified bases during transcription.49 The resulting modified RNA pool can be used for isolation of nuclease-stable RNA aptamers. Recently, a fully 2'-OCH3-modified anti-VEGF aptamer was selected, and when conjugated to 40 kDa PEG, it demonstrated a circulating half-life of 23 h.50 Conversely, a DNA polymerase that can incorporate unnatural bases such as 2'-F and 2'-OCH3 has not been described, and, consequently, DNA aptamers must be nuclease stabilized after the SELEX procedure.

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