Diels-Alder ribozyme

After ribozyme (100-mer) catalyzing Diels-Alder reaction was discovered in 1997 using the unique selection system, small Diels-Alder ribozyme (38-mer) with remarkable catalytic efficiency (20 000 relative to the uncatalyzed reaction) was developed. Recently, the novel ribozyme that catalyzed [4 - - 2] cycloaddition between tethered diene and biotinylated maleimide (Scheme 20) and the crystal structure of Diels-Alder ribozyme in the unbound form and in complex with a reaction product has been solved. ... 

Hyperfine spectroscopy methods, such as ID ESEEM, 2D HYSCORE, and ENDOR, have been employed to determine the coordination sphere of Mn " ions bound to the HHRz or to the Diels-Alder ribozyme. The HHRz was first investigated by X-band stimulated echo ESEEM by Britt and coworkers [102]. The ESEEM data revealed nitrogen hyperfine coupling of 2.3 MHz,... 

A. Serganov, S. Keiper, L. Malinina, V. Tereshko, E. Skripkin, C. Hobartner, A. Polonskaia, A. T. Phan, R. Wombacher, R. Micura, Z. Danter, A. Jaschke, D. J. Patel, Structural basis for Diels-Alder ribozyme-catalysed carbon-carbon bond formation, Nat. Struct. Mol. Biol., 2005, 12, 218-224. 

Cadmium(II)-induced EPR silencing was applied to evaluate the affinity of Mn " to several distinct binding sites in the Diels-Alder ribozyme [288]. As Cd " is... 

In addition, it has been discovered that there are naturally occurring enzymes that facilitate Diels-Alder type reactions within certain metabolic pathways and that enzymes are also instrumental in forming polyketides, isoprenoids, phenylpropanoids, and alkaloids (de Araujo et al., 2006). Agresti et al. (2005) identified ribozymes from RNA oligo libraries that catalyzed multiple-turnover Diels-Alder cycloaddition reactions. 

Agresti, J.J., Kelly, B.T., Jaschke, A., and Griffiths, A.D. (2005) Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc. Natl. Acad. Sci. USA 102,16170-16175. 

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]. 

Fig. 5.3.6. HPLC analysis of Diels-Alder reactions catalyzed by 49nt d-RNA and the enantiomeric 49nt L-ribozyme. Samples were analyzed on a chiral column with UV detection...

There is only one other example of ribozyme-catalyzed carbon-carbon bond formation, and this also is a Diels-Alder reaction [13, 14]. The ribozymes isolated by the Eaton group accelerated the reaction of a tethered aliphatic diene with biotinylated maleimide approximately 800-fold, although no activity was observed toward the two free reactants. A second feature is that these ribozymes contain modified nucleotides and their activity depends on the presence of cupric ion. 

The Houk group recently compared several artificial catalytic Diels-Alderase systems, including the ribozymes described here [11]. This study came to the conclusion that in none of these artificial systems there is a significant specific stabilization of the transition state. Acceleration arises predominantly from binding of the reactants, converting a second-order reaction of diene with dienophile into a first-order reaction of the termolecular complex of host, diene, and dienophile. The simultaneous presence of the two components of an intermolecular Diels-Alder reaction within the confined space of a cavity is the driving force that facilitates the reaction. 

Ribozyme-catalyzed reactions involving C-C bond formations have also been reported. Seelig and Jaschke (233) presented the in vitro selection of ribozyme catalysts for the Diels-Alder reaction between maleimide and anthracene, employing a 2 X lO -member library of 160-mer modified ONs (L28) with 120 randomized positions. The selection strategy used is shown in Fig. 10.40. Library L28 was prepared from the corresponding dsDNA sequences, and transcription initiation was performed in the presence of ternary complexes between guanosine monophosphate (10.57), PEG (10.58), and anthracene (10.59, step a. Fig. 10.40). The library obtained contained a 5 -anthracene-PEG appendage and was incubated with biotin-modified maleimide... 

Figure 10.40 Selection of artificial ribozymes with catalytic properties for a Diels-Alder reaction from the biosynthetic ON modified ribozyme library L28 library synthesis and the selection process using the biotinylated selection dienophile 10.60.

Nucleic acid selection methods have also been exploited for the development of novel RNA enzymes or ribozymes (58). An m-vitro-selected RNA that contains the modified nucleotide 5-(4-pyridylmethyl)-uridine (Table 1) can catalyze carbon-carbon bond formation in a Diels-Alder cycloaddition, with an 800-fold rate acceleration compared with a random RNA (49). Modified RNAs that contain the same uridine modification have also been selected to mediate metal-metal bond formation in the synthesis of palladium nanoparticles (59). Modified RNAs are likely to have many other applications as novel ribozymes that catalyze important biological reactions or can be used to create novel materials. 

Figure 14,4 Ribozyme catalysis of a Diels-Alder reaction, a Chemistry of the reaction, b Chiral HPLC analysis of products obtained in reactions either without catalyst (background reaction), with the catalytic 49mer minimum ribozyme or its synthetic mirror-image L-RNA version (R, = (C2H40)e-H R2 = (CH2)sCOOCH3) ...

Dotted lines represent pseudoknot Watson-Crick type interactions, b) Tertiary fold and c) three-dimensional topology in the crystal structure of the ribozyme - product complex. One enantiomer of the Diels-Alder product (sticks with transparent spheres) is bound into the catalytic pocket of the ribozyme ... 

---