Library aptamer

Keywords Ribozymes, In vitro selection. Nucleic acid libraries, Metallo enzymes, Aptamers. 

Direct Selections from Aptamer-Based Libraries. 

The fact that, in both selection experiments, new solutions regarding the structure of the functional molecules have been adopted demonstrates that the best sequence for binding is not necessarily the best sequence for performing catalysis. It seems likely that many of the sequence solutions could also have been selected from completely randomized pools. This notion is confirmed by the aforementioned study by Hager and Szostak [82], in which the mutated ATP-aptamer motif was also included in the starting library but where the resulting ribozyme had no relationship to the parent ATP-binding motif. 

Similarly, the authors also examined the stabilization effect of dynamic modification of a U-NH -appended RNA aptamer that forms a kissing complex with the HIVl transactivation-responsive RNA element TAR. In this dynamic library, 2-chloro-6-methoxy-3-quinofinecarboxaldehyde (Rd) was incorporated in place of benzaldehyde (Ra). After equilibration of the U-NHj-substituted aptamer and aldehydes Rb-Rd in the presence of the TAR RNA target, it was found that the nalidixic aldehyde Rc-appended RNA was amplified 20%, and accompanied by an increased (Fig. 3.17). Interestingly, the nalidixic aldehyde Rc was selected in both DNA and RNA complexation experiments. 

Figure 3.17 Approach to the dynamic combinatorial modification of the TAR-binding aptamer. Left, italics The TAR RNA sequence. Left, bold The TAR-binding aptamer. Left, boxed The 2 -amino-2-deoxyuridine (U-NH ) for dynamic RNA modification. Left center Rb—Rd, the aldehyde library components. Right center Imino-linked DCL members. Right The selected nalidixic aldehyde appended to U-NH results in the TAR RNA-aptamer complex stabilization.

In the DCC SELEX screen, aldehydes, the TAR RNA target, and a random library of 2 -amino RNAs were allowed to equilibrate. Next, the TAR RNA target and bound ligands were separated from the aptamer library. The selected 2 -amino RNAs that bound the TAR RNA target were then reverse transcribed into DNA and PCR amplified. These double-stranded... 

Figure 3.19 Schematic of the DCC SELEX system. Upper left A library of random 2 -amino RNAs are allowed to equilibrate via imine formation with aldehydes in the presence of target. Bottom left Modified RNAs are bound to the target. Bottom center Modified RNAs bound to the target are separated from unbound RNAs. Bottom right Selected RNAs are eluted and reverse transcribed and amplified to corresponding double-stranded DNA. Upper right The selected double-stranded DNA is transcribed to the 2 -amino RNAs. The selection process is repeated n-cycles and selected conjugated aptamers are identified.

Probes can be antibodies, other binding proteins constructed from protein fusions, or even oligonucleotide aptamers. While completion of the Human Genome Project has enabled access to content for nuclide acid arrays, the content for protein arrays is largely based upon available antibody libraries. Thus, the commercialization of protein microarrays remains largely dependent upon both commercial and institutional providers of protein content. These providers must also permit access to the data-based protein annotations. These are necessary in order for the protein array to be useful as a bioinformatics tool. 

By similar logic, protein affinity libraries have been constructed to identify protein—protein combining sites, as in antibody—antigen interaction (19) and recombinant libraries have been made which produce a repertoire of antibodies in E. coli (20). In another case, a potential DNA-based therapeutic strategy has been studied (21). DNAs from a partially randomized library were sdected to bind thrombin in vitro. Oligonucleotides, termed aptamers that bound thrombin shared a conserved sequence 14—17 nudeotides long. 

Combinatorial libraries are limited by the number of sequences that can be synthesized. For example, a library consisting of one molecule each of a 60-nudeotide sequence randomized at each position, would have a mass of >1014 g, well beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonudeotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete libraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (Kd) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constructing a less complex library for further selection. 

Aptamers are nucleic acids which exhibit a defined structure due to their nucleotide sequence and therefore, are able to specifically bind selected targets [1] (aptus [lat.] = fitting, sticking to). Aptamers and likewise, ribozymes [2] and deoxyribozymes [3] are selected in vitro by screening nucleic acid libraries. Here we describe in detail the selection of aptamers by a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment) [4]. 

Davis, J.H. and Szostak, J.W. (2002) Isolation of high-affinity GTP aptamers from partially structured RNA libraries, Proc. Natl. Acad. Sci. USA 99, 11616-11621. 

A single-stranded DNA library was also screened against the HIV-1 RT. IQ values for the selected DNA sequences were in the nanomolar range and they inhibited the DNA polymerase activity of this enzyme with a K, as low as 1 nM (Schneider et al., 1995). The best DNA aptamer folds as a hairpin with an internal loop and competes with the RNA pseudo-knot for RT binding. These two ligands share very little structural similarity. 

Famulok, M. and Jenne, A. (1998) Oligonucleotide libraries-variatio delectat. Curr. Opin. Chem. Biol., 2, 320-327. Famulok, M. and Mayer, G. (1999) Aptamers as tools in molecular biology and immunology. Curr. Top. Microbiol. Immunol., 243, 123-136. 

---