User:Kaguya-Taketori/脫氧核酶

  提示：此用户页的主题不是DNA酶。

脫氧核酶（Deoxyribozymes）係一類有催化作用的寡聚脫氧核苷酸。其催化作用與化學本質爲蛋白質的酶以及核酶（有催化作用的RNA）相似。科學家早已發現了大量的化學本質爲蛋白質的酶，核酶也在1980年代被發現 ，但截至2014年，仍無天然的脫氧核酶。值得注意的是，不能把脫氧核酶與DNA適體混爲一談。DNA適體的化學本質爲與配體相連的寡聚脫氧核苷酸，它不具有催化化學反應的能力。這是由核酸的官能團的缺乏造成的。相比擁有20種基本單位（氨基酸）的蛋白質，一種核酸只由四種基本單位（核苷酸）組成，且四種核苷酸的不同部分，核鹼基之間差異亦不明顯。DNA也沒有RNA獨有的2'-羥基（—OH），因而催化潛能進一步弱化。

除了本身催化潛能的欠缺外，自然脫氧核酶的缺乏亦可能與DNA的雙螺旋結構有關。雙螺旋結構不僅限制了DNA的物理柔韌性，還使得DNA難以形成三級結構，使得雙鏈DNA的催化潛能相當低。雖然生物界也中存在少數單鏈DNA，比如多拷貝單鏈DNA（msDNA），單鏈DNA病毒的單鏈DNA，以及DNA複製叉區域的單鏈DNA。另外，DNA和RNA的其他一些結構上的區別也造成了脫氧核酶的缺乏，比如DNA的胸腺嘧啶可被甲基化，這和RNA的尿嘧啶的情況形成了對比。另外，DNA更傾向於形成B型螺旋，而RNA更傾向於形成A型螺旋。然而，DNA亦能形成一些RNA不具有的結構，這說明儘管DNA和RNA存在一些結構上的差異，但它們的催化潛能並不會因爲它們的它們可能的結構基序而增加或減少。

類型

核糖核酸酶（RNA酶）

17E脫氧核酶的反式結構。大部分化學本質爲DNA的核糖核酸酶有相似的結構，由一條單獨的酶鏈（圖中用藍色和青色標出）和一條與酶鏈互補的襯底鏈（圖中用黑色標出）。互補鹼基形成的兩個臂位於催化核心的側翼（圖中用青色標出），有一個核糖核苷酸殘基位於襯底鏈上（圖中用紅色標出）。箭頭代表脫氧核酶的切割位點

種類最豐富的脫氧核酶爲核糖核酸酶（RNA酶）。這類核酶能通過酯交換反應切除一個核糖核苷酸殘基的磷酸二酯鍵，生成一個2'3'端環形磷酸基團末端和一個5'端羥基末端。脫氧核酶類RNA酶的酶切位點通常是長單鏈DNA上的一個單核糖核苷酸殘基。一旦確定了目標，單鏈的「順式」脫氧核酶就會隔開襯底鏈（包含單個核糖核苷酸殘基的那條鏈）的結構域和酶結構域（包含催化核心的區域），轉化爲雙鏈的「反式」脫氧核酶。被分開的襯底鏈和催化鏈可以通過催化核心的兩個含有互補鹼基的側翼臂雜交。1994年，在斯克里普斯研究所的杰拉爾德·喬伊斯手下做博士後的羅納德·布萊克是脫氧核酶的發現者。他發現的那種核酶即爲核糖核酸酶。這種脫氧核酶隨後被命名爲GR-5，它是一種Pb²⁺離子依賴性的酶，能以高於非催化條件下100倍的速率切除單核糖核酸的磷酸酯鍵。 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.

RNA ligases

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.

Other reactions

Many other deoxyribozymes have since been developed that catalyze DNA phosphorylation, DNA adenylation, DNA deglycosylation, porphyrin metalation, thymine dimer photoreversion and DNA cleavage.

Methods

主条目：Systematic evolution of ligands by exponential enrichment

in vitro selection

Because there are no known naturally occurring deoxyribozymes, most known deoxyribozyme sequences have been discovered through a high-throughput in vitro selection technique, similar to SELEX. in vitro selection utilizes a "pool" of a large number of random DNA sequences (typically 10¹⁴–10¹⁵ unique strands) that can be screened for a specific catalytic activity. The pool is synthesized through solid phase synthesis such that each strand has two constant regions (primer binding sites for PCR amplification) flanking a random region of a certain length, typically 25–50 bases long. Thus the total number of unique strands, called the sequence space, is 4^N where N denotes the number of bases in the random region. Because 4²⁵ ≈ 10¹⁵, there is no practical reason to choose random regions of less than 25 bases in length, while going above this number of bases means that the total sequence space cannot be surveyed. However, since there are likely many potential candidates for a given catalytic reaction within the sequence space, random regions of 50 and even higher have successfully yielded catalytic deoxyribozymes.

The pool is first subjected to a selection step, during which the catalytic strands are separated from the non-catalytic strands. The exact separation method will depend on the reaction being catalyzed. As an example, the separation step for ribonucleotide cleavage often utilizes affinity chromatography, in which a biological tag attached to each DNA strand is removed from any catalytically active strands via cleavage of a ribonucleotide base. This allows the catalytic strands to be separated by a column that specifically binds the tag, since the non-active strands will remain bound to the column while the active strands (which no longer possess the tag) flow through. A common set-up for this is a biotin tag with a streptavidin affinity column. Gel electrophoresis based separation can also be used in which the change in molecular weight of strands upon the cleavage reaction is enough to cause a shift in the location of the reactive strands on the gel. After the selection step, the reactive pool is amplified via Polymerase Chain Reaction (PCR) to regenerate and amplifiy the reactive strands, and the process is repeated until a pool of sufficient reactivity is obtained. Multiple rounds of selection are required because some non-catalytic strands will inevitably make it through any single selection step. Usually 4–10 rounds are required for unambiguous catalytic activity, though more rounds are often necessary for more stringent catalytic conditions. After a sufficient number of rounds, the final pool is sequenced and the individual strands are tested for their catalytic activity.

Deoxyribozymes obtained through in vitro selection will be optimized for the conditions during the selection, such as salt concentration, pH, and the presence of cofactors. Because of this, catalytic activity only in the presence of specific cofactors or other conditions can be achieved using positive selection steps, as well as negative selection steps against other undesired conditions.

in vitro evolution

A similar method of obtaining new deoxyribozymes is through in vitro evolution. Though this term is often used interchangeably with in vitro selection, in vitro evolution more appropriately refers to a slightly different procedure in which the initial oligonucleotide pool is genetically altered over subsequent rounds through genetic recombination or through point mutations. For point mutations, the pool can be amplified using error-prone PCR to produce many different strands of various random, single mutations. As with in vitro selection, the evolved strands with increased activity will tend to dominate the pool after multiple selection steps, and once a sufficient catalytic activity is reached, the pool can be sequenced to identify the most active strands.

The initial pool for in vitro evolution can be derived from a narrowed subset of sequence space, such as a certain round of an in vitro selection experiment, which is sometimes also called in vitro reselection. The initial pool can also be derived from amplification of a single oligonucleotide strand. As an example of the latter, a recent study showed that a functional deoxyribozyme can 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 first successful evolution of catalytic DNA from a non-catalytic precursor could provide support for the RNA World hypothesis. In another recent study, 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, though the researches hypothesized that the activity could be further increased through further selection. This first evidence for transfer of function between different nucleic acids could provide support for various pre-RNA World hypotheses.

"True" catalysis?

Because most deoxyribozymes suffer from product inhibition and thus exhibit single-turnover behavior, it is sometimes argued that deoxyribozymes do not exhibit "true" catalytic behavior since they cannot undergo multiple-turnover catalysis like most biological enzymes. However, the general definition of a catalyst requires only that the substance speeds up the rate of a chemical reaction without being consumed by the reaction (i.e. it is not permanently chemically altered and can be recycled). Thus, by this definition, single-turnover deoxyribozymes are indeed catalysts. Furthermore, many endogenous enzymes (both proteins and ribozymes) also exhibit single-turnover behavior, and so the exclusion of deoxyribozymes from the rank of "catalyst" simply because it does not feature multiple-turnover behavior seems unjustified.

Applications

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.

Sensors

DNAzymes have found practical use in metal biosensors.

Asymmetric synthesis

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.

Other uses

Other uses of DNA in chemistry are in DNA-templated synthesis, Enantioselective catalysis, DNA nanowires and DNA computing.

See also

• SELEX
• Ribozyme
• Aptamer

External links

• 醫學主題詞表（MeSH）：Deoxyribozyme