跳转到内容

用户:Kaguya-Taketori/脱氧核酶

维基百科，自由的百科全书
提示：此用户页的主题不是DNA酶。

脱氧核酶（Deoxyribozymes）系一类有催化作用的寡聚脱氧核苷酸。其催化作用与化学本质为蛋白质的酶以及核酶（有催化作用的RNA）相似[1]。科学家早已发现了大量的化学本质为蛋白质的酶，核酶也在1980年代被发现[2][3] ，但截至2014年，仍无天然的脱氧核酶[4]。值得注意的是，不能把脱氧核酶与DNA适体混为一谈。DNA适体的化学本质为与配体相连的寡聚脱氧核苷酸，它不具有催化化学反应的能力。这是由核酸的官能团的缺乏造成的。相比拥有20种基本单位（氨基酸）的蛋白质，一种核酸只由四种基本单位（核苷酸）组成，且四种核苷酸的不同部分，核碱基之间差异亦不明显。DNA也没有RNA独有的2'-羟基（—OH），因而催化潜能进一步弱化[5]。

除了本身催化潜能的欠缺外，自然脱氧核酶的缺乏亦可能与DNA的双螺旋结构有关。双螺旋结构不仅限制了DNA的物理柔韧性，还使得DNA难以形成三级结构（英语：Nucleic acid tertiary structure），使得双链DNA的催化潜能相当低[5]。虽然生物界也中存在少数单链DNA，比如多拷贝单链DNA（英语：multicopy single-stranded DNA）（msDNA），单链DNA病毒的单链DNA，以及DNA复制叉区域的单链DNA。另外，DNA和RNA的其他一些结构上的区别也造成了脱氧核酶的缺乏，比如DNA的胸腺嘧啶可被甲基化，这和RNA的尿嘧啶的情况形成了对比。另外，DNA更倾向于形成B型螺旋，而RNA更倾向于形成A型螺旋[1]。然而，DNA亦能形成一些RNA不具有的结构，这说明尽管DNA和RNA存在一些结构上的差异，但它们的催化潜能并不会因为它们的它们可能的结构基序而增加或减少[1]。

类型

核糖核酸酶（RNA酶）

17E脱氧核酶的反式结构。大部分化学本质为DNA的核糖核酸酶有相似的结构，由一条单独的酶链（图中用蓝色和青色标出）和一条与酶链互补的衬底链（图中用黑色标出）。互补碱基形成的两个臂位于催化核心的侧翼（图中用青色标出），有一个核糖核苷酸残基位于衬底链上（图中用红色标出）。箭头代表脱氧核酶的切割位点

种类最丰富的脱氧核酶为核糖核酸酶（RNA酶）。这类核酶能通过酯交换反应切除一个核糖核苷酸残基的磷酸二酯键，生成一个2'3'端环形磷酸基团末端和一个5'端羟基末端[5][6]。脱氧核酶类RNA酶的酶切位点通常是长单链DNA上的一个单核糖核苷酸残基。一旦确定了目标，单链的“顺式”脱氧核酶就会隔开衬底链（包含单个核糖核苷酸残基的那条链）的结构域和酶结构域（包含催化核心的区域），转化为双链的“反式”脱氧核酶。被分开的衬底链和催化链可以通过催化核心的两个含有互补碱基的侧翼臂杂交。1994年，在斯克里普斯研究所的杰拉尔德·乔伊斯手下做博士后的罗纳德·布莱克是脱氧核酶的发现者。他发现的那种核酶即为核糖核酸酶[7]。这种脱氧核酶随后被命名为GR-5[8]，它是一种Pb2+离子依赖性的酶，能以高于非催化条件下100倍的速率切除单核糖核酸的磷酸酯键[7]。 Subsequently, additional RNA-cleaving deoxyribozymes that incorporate different metal cofactors were developed, including the Mg2+-dependent E2 deoxyribozyme[9] and the Ca2+-dependent Mg5 deoxyribozyme.[10] 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.[11] 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."[6][12]

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,[13] UO22+-specific 39E,[14] and Na+-specific A43.[15]

RNA ligases

Of particular interest are DNA ligases.[5] 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[16] 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.[17][18] 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 4N 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.[18]

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.[17][18] 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.[18] 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,[6] 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.[18]

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.[17][18] 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.[18] 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.[19] 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.[20] 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.[5] Furthermore, many endogenous enzymes (both proteins and ribozymes) also exhibit single-turnover behavior,[5] 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.[21]

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.[22] 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,[23] DNA nanowires and DNA computing.[24]

See also

References

  1. ^ 1.0 1.1 1.2 Breaker, Ronald R. DNA enzymes. Nature Biotechnology (PDF). May 1997, 15 (5): 427–431. PMID 9131619. doi:10.1038/nbt0597-427. 
  2. ^ Kruger, Kelly; Grabowski, Paula J.; Zaug, Arthur J.; Sands, Julie; Gottschling, Daniel E.; Cech, Thomas R. Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell. November 1982, 31 (1): 147–157 [2015-07-10]. ISSN 0092-8674. PMID 6297745. doi:10.1016/0092-8674(82)90414-7. 
  3. ^ Guerrier-Takada, Cecilia; Gardiner, Katheleen; Marsh, Terry; Pace, Norman; Altman, Sidney. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. December 1983, 35 (3, Part 2): 849–857 [2015-07-10]. ISSN 0092-8674. doi:10.1016/0092-8674(83)90117-4. 
  4. ^ Breaker, Ronald R.; Joyce, Gerald F. The Expanding View of RNA and DNA Function. Chemistry & Biology. 2014-09-18, 21 (9): 1059–1065 [2015-07-09]. ISSN 1074-5521. doi:10.1016/j.chembiol.2014.07.008. 
  5. ^ 5.0 5.1 5.2 5.3 5.4 5.5 Silverman, Scott K. Deoxyribozymes: DNA catalysts for bioorganic chemistry (PDF). Org. Biomol. Chem. 2004, 2 (19): 2701–06. ISSN 1477-0539. PMID 15455136. doi:10.1039/B411910J. 
  6. ^ 6.0 6.1 6.2 Silverman, Scott K. In vitro selection, characterization, and application of deoxyribozymes that cleave RNA. Nucleic Acids Research. 2005, 33 (19): 6151–6163 [2015-07-15]. ISSN 0305-1048. PMC 1283523 可免费查阅. PMID 16286368. doi:10.1093/nar/gki930. 
  7. ^ 7.0 7.1 Breaker, Ronald R.; Joyce, Gerald F. A DNA enzyme that cleaves RNA. Chem Biol. December 1994, 1 (4): 223–229. PMID 9383394. doi:10.1016/1074-5521(94)90014-0. 
  8. ^ Lan, Tian; Furuya, Kimberly; Lu, Yi. A highly selective lead sensor based on a classic lead DNAzyme. Chemical Communications. 2010-06-14, 46 (22): 3896–3898 [2015-07-30]. ISSN 1364-548X. PMID 20407665. doi:10.1039/B926910J. 
  9. ^ Breaker, Ronald R.; Joyce, Gerald F. A DNA enzyme with Mg2+-dependent RNA phosphoesterase activity. Chemistry & Biology. 1995-01-10, 2 (10): 655–660 [2015-07-16]. ISSN 1074-5521. PMID 9383471. doi:10.1016/1074-5521(95)90028-4. 
  10. ^ Faulhammer, Dirk; Famulok, Michael. The Ca2+ Ion as a Cofactor for a Novel RNA-Cleaving Deoxyribozyme. Angewandte Chemie International Edition in English. 1996-12-01, 35 (23–24): 2837–2841 [2015-07-17]. ISSN 1521-3773. doi:10.1002/anie.199628371. 
  11. ^ Santoro, Stephen W.; Joyce, Gerald F. A general purpose RNA-cleaving DNA enzyme. Proceedings of the National Academy of Sciences. 1997-04-29, 94 (9): 4262–4266 [2015-07-17]. ISSN 0027-8424. PMID 9113977. doi:10.1073/pnas.94.9.4262. 
  12. ^ Cruz, Rani P. G; Withers, Johanna B; Li, Yingfu. Dinucleotide Junction Cleavage Versatility of 8-17 Deoxyribozyme. Chemistry & Biology. January 2004, 11 (1): 57–67 [2015-08-13]. ISSN 1074-5521. PMID 15112995. doi:10.1016/j.chembiol.2003.12.012. 
  13. ^ Li, Jing; Lu, Yi. A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions. Journal of the American Chemical Society. 2000-10-01, 122 (42): 10466–10467 [2015-05-17]. ISSN 0002-7863. doi:10.1021/ja0021316. 
  14. ^ Wu, Peiwen; Hwang, Kevin; Lan, Tian; Lu, Yi. A DNAzyme-Gold Nanoparticle Probe for Uranyl Ion in Living Cells. Journal of the American Chemical Society. 2013-04-10, 135 (14): 5254–5257. ISSN 0002-7863. PMC 3644223 可免费查阅. PMID 23531046. doi:10.1021/ja400150v. 
  15. ^ Torabi, Seyed-Fakhreddin; Wu, Peiwen; McGhee, Claire E.; Chen, Lu; Hwang, Kevin; Zheng, Nan; Cheng, Jianjun; Lu, Yi. In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing. Proceedings of the National Academy of Sciences. 2015-05-12, 112 (19): 5903–5908 [2015-05-17]. ISSN 0027-8424. PMID 25918425. doi:10.1073/pnas.1420361112. 
  16. ^ Daniel J.-F. Chinnapen; Dipankar Sen. A deoxyribozyme that harnesses light to repair thymine dimers in DNA. US National Library of Medicine: Proceedings of the National Academy of Sciences of the United States of America. Jan 6, 2004. PMID 14691255. 
  17. ^ 17.0 17.1 17.2 Joyce, Gerald F. Directed Evolution of Nucleic Acid Enzymes. Annual Review of Biochemistry. 2004, 73 (1): 791–836 [2015-07-27]. PMID 15189159. doi:10.1146/annurev.biochem.73.011303.073717. 
  18. ^ 18.0 18.1 18.2 18.3 18.4 18.5 18.6 Silverman, Scott K. Catalytic DNA (deoxyribozymes) for synthetic applications—current abilities and future prospects. Chemical Communications. 2008-07-23, (30): 3467–3485 [2015-07-27]. ISSN 1364-548X. doi:10.1039/B807292M. 
  19. ^ Gysbers, Rachel; Tram, Kha; Gu, Jimmy; Li, Yingfu. Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence. Scientific Reports. 2015-06-19, 5: 11405 [2015-07-15]. PMC 4473686 可免费查阅. PMID 26091540. doi:10.1038/srep11405. 
  20. ^ Paul, Natasha; Springsteen, Greg; Joyce, Gerald F. Conversion of a Ribozyme to a Deoxyribozyme through In Vitro Evolution. Chemistry & Biology. March 2006, 13 (3): 329–338 [2015-07-20]. ISSN 1074-5521. PMID 16638538. doi:10.1016/j.chembiol.2006.01.007. 
  21. ^ Juewen Liu; Yi Lu. Optimization of a Pb2+-Directed Gold Nanoparticle/DNAzyme Assembly and Its Application as a Colorimetric Biosensor for Pb2+. Chem. Mater. 2004, 16 (17): 3231–38. doi:10.1021/cm049453j. 
  22. ^ Gerard Roelfes; Ben L. Feringa. DNA-Based Asymmetric Catalysis. Angewandte Chemie International Edition. 2005, 44 (21): 3230–2. PMID 15844122. doi:10.1002/anie.200500298. 
  23. ^ García-Fernández, Almudena; Roelfez, Gerard. Chapter 9. Enantioselective catalysis at the DNA Scaffold. Astrid Sigel, Helmut Sigel and Roland K. O. Sigel (编). Interplay between Metal Ions and Nucleic Acids. Metal Ions in Life Sciences 10. Springer. 2012: 249–268. doi:10.1007/978-94-007-2172-2_9. 
  24. ^ Yoshihiro Ito; Eiichiro Fukusaki. DNA as a 'Nanomaterial' (PDF). Journal of Molecular Catalysis B: Enzymatic. 2004, 28 (4–6): 155–166. doi:10.1016/j.molcatb.2004.01.016. 
检索自“https://zh.wikipedia.org/w/index.php?title=User:Kaguya-Taketori/脫氧核酶&oldid=52647672”