Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are DNA oligonucleotides that are capable of catalyzing specific chemical reactions, similar to the action of other biological enzymes, such as proteins or ribozymes (enzymes composed of RNA). However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s, there are no known naturally occurring deoxyribozymes.
With the exception of ribozymes, nucleic acid molecules within cells primarily serve as storage of genetic information due to their ability to form base pairs, which allows for high-fidelity copying and transfer of genetic information. In contrast, nucleic acid molecules which act as enzymes are limited in their catalytic ability to hydrogen bonding, pi stacking, and metal-ion coordination. This is because, unlike proteins which are built from up to twenty different amino acids with various functional groups, nucleic acids are built from just four relatively similar nucleobases. In addition, DNA lacks the 2'-hydroxyl group found in RNA, which further limits the catalytic competency of deoxyribozymes in comparison to ribozymes. The lack of naturally occurring deoxyribozymes may also be due to the primarily double-stranded conformation of DNA in biological systems which would limit its physical flexibility and ability to form tertiary structures, and so would drastically limit the ability of double-stranded DNA to act as a catalyst; though there is at least one known instance of biological single-stranded DNA: multicopy single-stranded DNA (msDNA). Further structural differences between DNA and RNA may also play a role in the lack of biological deoxyribozymes, such as the additional methyl group of the DNA base thymidine compared to the RNA base uracil or the tendency of DNA to adopt the B-form helix while RNA tends to adopt the A-form helix. However, it has also been shown that DNA can form structures that RNA cannot, which suggests that, though there are differences in structures that each can form, neither is inherently more or less catalytic due to their possible structural motifs.
The most abundant class of deoxyribozymes are ribonucleases, which catalyze the cleavage of a ribonucleotide phosphodiester bond through a transesterification reaction, forming a 2'3'-cyclic phosphate terminus and a 5'-hydroxyl terminus. Ribonuclease deoxyribozymes typically undergo selection as long, single-stranded oligonucleotides which contain a single ribonucleotide base to act as the cleavage site. Once sequenced, this single-stranded "cis"-form of the deoxyribozyme can be converted to the two-stranded "trans"-form by separating the substrate domain (containing the ribonucleotide cleavage site) and the enzyme domain (containing the catalytic core) into separate strands which can hybridize through two flanking arms consisting of complementary base pairs.
The first known deoxyribozyme was a ribonuclease, discovered in 1994 by Ronald Breaker while a postdoctoral fellow in the laboratory of Gerald Joyce at the Scripps Research Institute. This deoxyribozyme catalyzes the Pb2+-dependent cleavage of a single ribonucleotide phosphoester at a rate that is more than 100-fold compared to the uncatalyzed reaction. Subsequently, additional RNA-cleaving deoxyribozymes that incorporate different metal cofactors were developed, including the Mg2+-dependent E2 deoxyribozyme and the Ca2+-dependent Mg5 deoxyribozyme. These first deoxyribozymes were unable to catalyze a full RNA substrate strand, but by incorporating the full RNA substrate strand into the selection process, deoxyribozymes which functioned with substrates consisting of either full RNA or full DNA with a single RNA base were both able to be utilized. The first of these more versatile deoxyribozymes, 8-17 and 10-23, are currently the most widely studied deoxyribozymes. In fact, many subsequently discovered deoxyribozymes were found to contain the same catalytic core motif as 8-17, including the previously discovered Mg5, suggesting that this motif represents the "simplest solution for the RNA cleavage problem."
Other notable deoxyribozyme ribonucleases are those that are highly selective for a certain cofactor. Among this group are the metal selective deoxyribozymes such as Pb2+-specific 17E, UO22+-specific 39E, and Na+-specific A43.
Of particular interest are DNA ligases. These molecules have demonstrated remarkable chemoselectivity in RNA branching reactions. Although each repeating unit in a RNA strand owns a free hydroxyl group, the DNA ligase takes just one of them as a branching starting point. An accomplishment unattainable with traditional organic chemistry.
in vitro selection
Because there are no known naturally occurring deoxyribozymes, most currently known deoxyribozyme sequences have been discovered through a high-throughput in vitro selection technique, similar to SELEX. in vitro selection utilizes combinatorial chemistry to produce a "pool" consisting of a large number of random DNA sequences (up to 1016 unique strands) that can be screened for a specific catalytic activity. Multiple rounds of selection are typically required, during which non-catalytic strands are removed from the pool, while catalytically active strands are enriched through amplification via PCR. The large number of initial strands in the pool give a higher chance that a catalytically active strand will be identified. Catalytic activity only in the presence of specific cofactors can also be achieved, using positive and negative selection steps.
in vitro evolution
A similar method of obtaining new deoxyribozymes is through in vitro evolution. Unlike in vitro selection which starts with a large number of random DNA strands, in vitro evolution starts with a single strand of a known DNA sequence. The strand is amplified using error-prone PCR to produce many different strands with random point mutations. These derivative strands are then observed for increased catalytic activity. New strands with activity can typically be generated after multiple rounds of evolution, similar to the multiple rounds of selection of in vitro selection.
Many different types of starting DNA strands can be chosen from. For example, an RNA ligase ribozyme was converted into a deoxyribozyme through in vitro evolution of the inactive deoxyribo-analog of the ribozyme. The new RNA ligase deoxyribozyme contained just twelve point mutations, two of which had no effect on activity, and had a catalytic efficiency of approximately 1/10 of the original ribozyme. This first evidence for transfer of function between different nucleic acids could provide support for various pre-RNA World hypotheses. Another recent study showed that a functional deoxyribozyme can also be selected through in vitro evolution of a non-catalytic oligonucleotide precursor strand. An arbitrarily chosen DNA fragment derived from the mRNA transcript of bovine serum albumin was evolved through random point mutations over 25 rounds of selection. Through deep sequencing analysis of various pool generations, the evolution of the most catalytic deoxyribozyme strand could be tracked through each subsequent single mutation. This evidence that catalytic DNA could be evolved from a non-catalytic precursor could provide support for the RNA World hypothesis.
An inherent disadvantage of DNA enzymes is product inhibition and single-turnover behavior. It may therefore be argued if DNA enzymes can be counted as true catalysts. On the other hand, low catalytic turnover is observed with many natural (non-DNA) occurring enzymes.
Although the discovery of RNA enzymes predates that of DNA enzymes the latter have some distinct advantages. DNA has better cost-effectiveness and DNA can be made with longer sequence length and can be made with higher purity in Solid-phase synthesis.
DNAzymes have found practical use in metal biosensors.
Chirality is another property that a DNAzyme can exploit. DNA occurs in nature as a right-handed double helix and in asymmetric synthesis a chiral catalyst is a valuable tool in the synthesis of chiral molecules from an achiral source. In one application an artificial DNA catalyst was prepared by attaching a copper ion to it through a spacer. The copper - DNA complex catalysed a Diels-Alder reaction in water between cyclopentadiene and an aza chalcone. The reaction products (endo and exo) were found to be present in an enantiomeric excess of 50%. Later it was found that an enantiomeric excess of 99% could be induced, and that both the rate and the enantioselectivity were related to the DNA sequence.
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