RNA aptamer

Matsugami A, Kobayashi S, Ouhashi K, Uesugi S, Yamamoto R, Taira K, Nishikawa S, Kumar PK, Katahira M (2003) Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer. Structure 11 533-545... 

Bridonneau. R, Bunch, S., Tengler, R., Hill, K., Carter, J., Pieken, W., Tinner-meier, D., Lehrman, R., and Drolet, D. W., Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity, /. Chromatogr. B, 726, 237, 1999. 

Holeman LA, Robinson SL, Szostak JW, Wilson C (1998) Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des 3 423 -31... 

Gouda, H. Kuntz, I.D. Case, D.A. Kollman, P.A., Free energy calculations for theophylline binding to an RNA aptamer Comparison of MM-PBSA and thermodynamic integration methods, Biopolymers 2003, 68,16-34. 

Rusconi CP, Scardino E, Layzer J, Pitoc GA, Ortel TL, Monroe D, Sullenger BA (2002) RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419 90-94... 

Ulrich H, Trujillo CA, Nery AA, Alves JM, Majumder P, Resende RR, Martins AH (2006) DNA and RNA aptamers from tools for basic research towards therapeutic applications. Comb Chem Fligh Throughput Screen 9 619-632... 

The cyanine dye dimethylindole red (DIR), with significantly reduced affinity for double-stranded DNA and nonspecific RNA, was used to generate a fluorescent complex (fluoromodule) with high affinity RNA aptamers (Fig. 12) [8]. DIR has... 

Fig. 6.1 I mino region of proton ID spectrum of ATP-binding RNA aptamer complex consisting of 40 nucleotides and bound AMP.

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

Following the TSA-based strategy, RNA aptamers were selected that specifically complexed the TSA for the isomerization of an asymmetrically substituted biphenyl derivative (Scheme 1) [7]. The selection was performed by affinity chromatography of a randomized pool on the TSA immobilized on agarose. After seven rounds of selection, the RNA pool accelerated the basal reaction 100-fold and was completely inhibited by the planar TSA. 

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.

Figure 3.20 TAR RNA DCC SELEX system, employing 2 -ammo-2-deoxyuri-dine (U-NH ) capable of reversible imine formation with the appended aldehydes Rb, Rc, and Re. Selected appended RNA aptamers and their corresponding dissociation constants are shown at the bottom.

Bugaut, A. Toulme, J-J. Rayner, B. SELEX and dynamic combinatorial chemistry interplay for the selection of conjugated RNA aptamers. Org. Bio-mol. Chem. 2006, 4, 4082 088. 

Enrichment of high affinity candidates is usually achieved in 8 to 15 rounds of SELEX. Each rotmd takes approximately 2 days to perform. The process has been automated using robotic liquid handlers both for DNA (SomaLogic) and RNA aptamers (Cox, 2002). Next, the sequenced aptamer is prepared in bulk by conventional DNA synthesis chemistry and purified, then the aptamer arrayed onto a solid support. Thus, an aptamer is ready for application within 2 to 3 mo. Because the sequence is known, preparation of additional aptamer is easily accomplished using conventional oligonucleotide chemical synthesis. 

Structures of Neomycin and Tobramycin in Complex with RNA Aptamers... 

Key words RNA aptamers, SELEX, Nicotinic acetylcholine receptors... 

A simple protocol detailed in this chapter was established to develop RNA aptamers that bind to the electric organ nAChR and that are displaced by cocaine (8) (see Fig. 1 for a scheme). This protocol can be easily transferred to SELEX applications with other receptors or cell-surface epitopes, given that these are enriched in membrane preparations. 

Fig. 1. Scheme for RNA aptamer identification targeting cocaine-binding sites on the nicotinic acetylcholine receptor (nAchR). 

For the elution of receptor-bound, cocaine-displaceable RNA aptamers, incubate each of these filter pieces in 100 pi of 1 mM cocaine (or 100 pi of 1 mM MK-801 that binds to the same site as cocaine on the nAChR) in incubation buffer for 20 min at room temperature. 

Fig. 2. Alternation ot gel-shitt and filter-binding selection steps Target-bound and unbound radiolabeled RNA aptamers are separated by polyacrylamide gel electrophoresis, visualized by autoradiography, purified from the gel, and used for the subsequent nitrocellulose-filter binding selection step. The experiments are earned out in the presence (-i-) and absence (-) of target protein using the SELEX cycles 0 (control), 3, and 7. The figure illustrates the increase of binding affinity of selected RNA pools, seen as augmented quantity of RNA retained together with the receptor protein at the top of the gel (modified from ref. (8)).

Determination of unspecific binding reaction by competition of the RNA aptamers with cocaine 79 pi incubation buffer, 10 pi nAChR-enriched plasma membranes (800 pg/ml protein 1.6 pM receptor), 1 pi P-RNA dilution containing 10 mg/ml t-RNA anti-RNAse (40 U/pl), and 10 pi cocaine (10 mM). The percentage of binding of the ( P) RNA aptamers to the... 

For cloning and sequencing of individual RNA aptamers, nine SELEX cycles are necessary to obtain high-affinity RNA ligands for the nAChR. 

Amplify plasmid inserts coding for individual RNA aptamers by PGR using the primers P-40 and P-22pGEM and purify the PGR products on a low-melting agarose gel. 

Screening for biological activity The biological activity of the selected RNA aptamers are determined in vitro, as to whether they inhibit the nAChR function as cocaine does or whether they compete with cocaine but do not have any biological activity by themselves and, therefore, protect the receptor against inhibition by cocaine (25). 

Repeat the experiment with 100 pM carbamoylcholine in the presence of increasing concentrations of the RNA aptamer to be tested, in order to determine if the RNA aptamer inhibits receptor function. If an RNA aptamer does not inhibit receptor function, experiments are performed in the presence of a constant concentration of carbamoylcholine and cocaine with increasing concentrations of the RNA aptamer to determine whether the aptamer alleviates inhibition (25). 

---