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Combinatorial Approaches for Isolating Functional Nucleic Acid Foldamers

The great functional diversity of nucleic acids, exemplified in Section 10.2.2, essentially results from combinatorial techniques known as ''in vitro selection'', ''in vitro evolution'' or SELEX (Systematic Evolution of Ligands by Exponential enrichment) techniques (see the reviews [132-134]). It exploits the construction of large libraries of DNA or RNA sequences, with the possibility of amplifying a small subset of selected molecules by PCR or RT-PCR. An example of an in vitro selection experiment is presented in Fig. 10.8.

First, a large library of DNA molecules is synthesized from a pool of synthetic semi-randomized oligonucleotides that are amplified by PCR to generate multiple

Fig. 10.8 Example of a round of SELEX. (step 1) A random pool of RNA molecules is synthesized by in vitro run-off transcription of a random DNA library generated by PCR. After folding in the presence of salts (step 2), the resulting population of 2° and 3C foldamers is submitted to a selection criterion such as binding to a molecule target. The functional molecules can be separated from the inactive ones by a

Fig. 10.8 Example of a round of SELEX. (step 1) A random pool of RNA molecules is synthesized by in vitro run-off transcription of a random DNA library generated by PCR. After folding in the presence of salts (step 2), the resulting population of 2° and 3C foldamers is submitted to a selection criterion such as binding to a molecule target. The functional molecules can be separated from the inactive ones by a selection assay such as a gel shift or affinity column assay (step 3). The selected molecules are then amplified by reverse transcription (RT) (step 4) and PCR (step 5) and eventually resubmitted to an additional round of selection. Mutations can eventually be introduced during this amplification steps to improve the activity of the pool of functional molecules.

double-strand DNA copies. A library typically contains molecules with two invariable sequence regions at the 5' and 3' ends (for amplification purposes) bracketing a random region including up to 200 nucleotides. Initial DNA libraries can contain up to 1016 individual molecules [75]. For DNA selection, the double-stranded DNA library is first denatured so that only one DNA strand enters into the selection process. For RNA selection, the DNA library is first transcribed into RNA. The population of single-stranded molecules is then challenged to perform a specific task that can be recognition of a molecular target or catalysis of a specific chemical reaction. This selection step is the most critical of the whole SELEX process and requires efficient physical separation of the functional molecules from the nonfunctional ones. Once separated, the selected molecules are amplified by PCR or RT-PCR and subjected to additional rounds of selection-amplification until the functional activity of the population can be detected through biochemical assay in the pool of selected molecules. The selected molecules can then be cloned, sequenced and tested individually for function and eventually further optimized after introduction of mutations by partial randomization or mutagenic PCR [132-134].

This powerful and very versatile technique has recently been automated so that selection can be performed in a matter of days instead of weeks [135-141]. Another interesting method called continuous in vitro evolution offers the possibility of evolving self-modifying ribozymes over several hundreds of generations [132]. The applicability of this system is limited, however.

SELEX strategies can also be applied to nucleic acids analogs synthesized by enzymatic incorporation of modified nucleotides and amplifiable by polymerases variants (see previous section). Alternatively, when nucleic acid analogs might not be amplifiable by PCR or RT-PCR, new methods such as non-SELEX selection can potentially be used to isolate aptamers in only one round of selection [142]. This latter technique developed by analytical chemists is derived from capillary electrophoresis SELEX (CE-SELEX). Because of the higher partition coefficient of CE over more traditional chromatographic techniques, this method allows identification of aptamers with extremely well defined affinity profiles in a limited number of rounds of selection [143, 144]. Another technique of selection, initially developed for protein selection and evolution, has recently been adapted to the isolation of transacting ribozymes through in vitro compartmentalization [145147]. It is worth mentioning that aptamers can also be selected against heterogeneous mixtures of targets such as whole cells offering the possibility of discriminating against different cell types, even when specific biomarkers are not known in advance. This type of approach has been also reviewed recently [148].

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