Deoxyribozymes: selection design and serendipity in the development of DNA catalysts

Abstract

One of the chemist's key motivations is to explore the forefront of catalysis. In this Account, we describe our laboratory's efforts at one such forefront: the use of DNA as a catalyst. Natural biological catalysts include both protein enzymes and RNA enzymes (ribozymes), whereas nature apparently uses DNA solely for genetic information storage. Nevertheless, the chemical similarities between RNA and DNA naturally lead to laboratory examination of DNA as a catalyst, especially because DNA is more stable than RNA and is less costly and easier to synthesize. Many catalytically active DNA sequences (deoxyribozymes, also called DNAzymes) have been identified in the laboratory by in vitro selection, in which many random DNA sequences are evaluated in parallel to find those rare sequences that have a desired functional ability. Since 2001, our research group has pursued new deoxyribozymes for various chemical reactions. We consider DNA simply as a large biopolymer that can adopt intricate three-dimensional structure and, in the presence of appropriate metal ions, generate the chemical complexity required to achieve catalysis. Our initial efforts focused on deoxyribozymes that ligate two RNA substrates. In these studies, we used only substrates that are readily obtained biochemically. Highly active deoxyribozymes were identified, with emergent questions regarding chemical selectivity during RNA phosphodiester bond formation. Deoxyribozymes allow synthesis of interesting RNA products, such as branches and lariats, that are otherwise challenging to prepare. Our experiments have demonstrated that deoxyribozymes can have very high rate enhancements and chemical selectivities. We have also shown how the in vitro selection process itself can be directed toward desired goals, such as selective formation of native 3'-5' RNA linkages. A final lesson is that unanticipated selection outcomes can be very interesting, highlighting the importance of allowing such opportunities in future experiments. More recently, we have begun using nonoligonucleotide substrates in our efforts with deoxyribozymes. We have especially focused on developing DNA catalysts for reactions of small molecules or amino acid side chains. For example, new deoxyribozymes have the catalytic power to create a nucleopeptide linkage between a tyrosine or serine side chain and the 5'-terminus of an RNA strand. Although considerable further work remains to establish DNA as a practical catalyst for small molecules and full-length proteins, the progress to date is very promising. The many lessons learned during the experiments described in this Account will help us and others to realize the full catalytic power of DNA.

Figure 1

RNA substrate combinations for DNA-catalyzed RNA ligation. (A) 2′,3′-Cyclic phosphate + 5′-hydroxyl. (B) 3′-Hydroxyl + 5′-triphosphate.

Figure 2

General in vitro selection strategy to identify deoxyribozymes that ligate RNA. The indicated conditions in the key selection step B can be changed as desired.

Figure 3

Arrangements of DNA and RNA for DNA-catalyzed RNA ligation. Base pairs in the binding arms are not depicted quantitatively. (A) Detailed arrangement of the first RNA ligation experiments, using either substrate combination from Figure 1., (B) Subsequently used arrangement with the 5′-triphosphate substrate combination.

Figure 4

Formation of 2′,5′-branched RNA by attack of an internal 2′-OH group of L into the 5′-triphosphate of R.

Figure 5

Deoxyribozymes that form a three-helix-junction (3HJ) architecture with their RNA substrates. (A) The 7S11 deoxyribozyme, which forms 2′,5′-branched RNA., The related 10DM24 deoxyribozyme has a similar architecture. (B) Conversion of 10DM24 to use GTP as a small-molecule substrate along with a shortened oligonucleotide cofactor (R_Δ). (C) Use of two molecules of GTP as substrate and cofactor, along with an oligonucleotide cofactor (R_ΔΔ).

Figure 6

Two general deoxyribozymes for RNA ligation using the 5′-triphosphate substrate combination.

Figure 7

Strategy for deoxyribozyme-catalyzed labeling (DECAL) of an RNA target. The labeled tagging RNA is prepared (top) and attached to the target RNA using the 10DM24 deoxyribozyme (bottom).

Figure 8

Deoxyribozymes that catalyze the Diels-Alder reaction. (A) In vitro selection strategy. (B) Intermolecular catalysis of the reaction.

Figure 9

DNA-catalyzed nucleopeptide linkage formation between a tyrosine side chain and the 5′-triphosphate of an RNA oligonucleotide. The substrates are held by the Tyr1 deoxyribozyme in a 3HJ architecture (compare Figure 5).