RibozymeRibozymeRibozymeRibozyme 核酶 (Catalytic RNA molecule) (Catalytic RNA molecule) (Catalytic RNA molecule) (Catalytic RNA molecule)

制作：制作：制作：制作：cz12062008cz12062008cz12062008cz12062008

1.1.1.1.What is a Ribozyme?What is a Ribozyme?What is a Ribozyme?What is a Ribozyme?

Ribozymes are true enzymesRibozymes are true enzymesRibozymes are true enzymesRibozymes are true enzymes....

A ribozyme is an RNA moleculeA ribozyme is an RNA moleculeA ribozyme is an RNA moleculeA ribozyme is an RNA molecule（分子） that is capable that is capable that is capable that is capable（能力） of catalyzing of catalyzing of catalyzing of catalyzing （催化）a chemical a chemical a chemical a chemical reaction.reaction.reaction.reaction.NOT PROTEINNOT PROTEINNOT PROTEINNOT PROTEIN（蛋白）

First Ribozyme First Ribozyme First Ribozyme First Ribozyme discovereddiscovereddiscovereddiscovered

上世纪80808080年代，美国科罗拉多大学博尔德分校的Thomas CechThomas CechThomas CechThomas Cech（The intron inThe intron inThe intron inThe intron in the pre-rRNA ofthe pre-rRNA ofthe pre-rRNA ofthe pre-rRNA of Tetrahemena isTetrahemena isTetrahemena isTetrahemena is self-splicedself-splicedself-splicedself-spliced）和美国耶鲁大学的Sidnery AltaSidnery AltaSidnery AltaSidnery Altannnn（The M1 The M1 The M1 The M1 RNARNARNARNA in ribonucleasein ribonucleasein ribonucleasein ribonuclease P is catalyticP is catalyticP is catalyticP is catalytic）各自独立地发现RNARNARNARNA具有生物催化功能....从而改变了生物催比剂的传统概念。

1989 Nobel Prize1989 Nobel Prize1989 Nobel Prize1989 Nobel PrizeIn ChemistryIn ChemistryIn ChemistryIn Chemistry

Sid AltmanSid AltmanSid AltmanSid Altman Tom CechTom CechTom CechTom Cech

I型内含子剪接型核酶 II型内含子

锤头核酶剪切型核酶 发夹核酶 自体催化 丁型肝炎病毒（HDV)HDV)HDV)HDV)核酶 RNaseP RNaseP RNaseP RNaseP 异体催化

2.2.2.2.核酶的分类

通过既剪又接的方式除去内含子（Intron)Intron)Intron)Intron)

自身催化的反应是只切不接。异体催化能剪切所tRNAtRNAtRNAtRNA前体的5555‘‘‘‘端

How many ribozyme ? How many ribozyme ? How many ribozyme ? How many ribozyme ? the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus) the hammerhead ribozyme (plant virus)

锤头状核酶（来源：植物病毒）

- the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus)- the hairpin ribozyme (plan virus) 发夹状核酶

- hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus)- hepatitis delta ribozyme (human virus) 丁型肝炎核酶（来源：人类病毒）

- neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA)- neurospora VS ribozyme (mitochondrial RNA) 孢VSVSVSVS核酶（来源：线粒体RNARNARNARNA）

- group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA)- group I and group II intron ribozyme (rRNA and mt RNA) 内含子IIII和IIIIIIII核酶（来源：rRNArRNArRNArRNA和MT RNAMT RNAMT RNAMT RNA）

- RNAse P (tRNA maturation)- RNAse P (tRNA maturation)- RNAse P (tRNA maturation)- RNAse P (tRNA maturation) 核糖核酸酶PPPP（来源：tRNAtRNAtRNAtRNA的成熟）

- Ribosome (translation) - Ribosome (translation) - Ribosome (translation) - Ribosome (translation) 核糖体（翻译）

- Spliceosome (splicing)- Spliceosome (splicing)- Spliceosome (splicing)- Spliceosome (splicing) 剪接 （拼接）

RibozymRibozymRibozymRibozym SequencedSequencedSequencedSequenced Size (nt) Size (nt) Size (nt) Size (nt) Activity Activity Activity Activity SourceSourceSourceSource （核酶） （测序） （大小） （功能） （来源）Group I intronGroup I intronGroup I intronGroup I intron >1>1>1>1’’’’500500500500 210 210 210 210 self-splicing via 3self-splicing via 3self-splicing via 3self-splicing via 3’’’’-OH-OH-OH-OH eukayotic pre-mRNA,eukayotic pre-mRNA,eukayotic pre-mRNA,eukayotic pre-mRNA,

of G; transesterificationof G; transesterificationof G; transesterificationof G; transesterification organelles, few bacteria organelles, few bacteria organelles, few bacteria organelles, few bacteria

Group II intronGroup II intronGroup II intronGroup II intron >700 >700 >700 >700 500 500 500 500 self-splicing via 3 self-splicing via 3 self-splicing via 3 self-splicing via 3’’’’-OH -OH -OH -OH organelles, few prokaryotes organelles, few prokaryotes organelles, few prokaryotes organelles, few prokaryotes of A; transesterification of A; transesterification of A; transesterification of A; transesterification HammerheadHammerheadHammerheadHammerhead 11 11 11 11 40 40 40 40 self-cleavage via self-cleavage via self-cleavage via self-cleavage via plant viruses plant viruses plant viruses plant virusesHepatitis delta virus Hepatitis delta virus Hepatitis delta virus Hepatitis delta virus 2 2 2 2 90 transesterification 90 transesterification 90 transesterification 90 transesterification hepatitis B virus hepatitis B virus hepatitis B virus hepatitis B virus (RNAgen.)(RNAgen.)(RNAgen.)(RNAgen.)HairpinHairpinHairpinHairpin 1 1 1 1 70 (2 70 (2 70 (2 70 (2’’’’,3,3,3,3’’’’-cyclic phosphate)-cyclic phosphate)-cyclic phosphate)-cyclic phosphate) satellite RNA of plant virussatellite RNA of plant virussatellite RNA of plant virussatellite RNA of plant virus

RNAse PRNAse PRNAse PRNAse P >500 >500 >500 >500 300 300 300 300 pre-tRNA processingpre-tRNA processingpre-tRNA processingpre-tRNA processing prokaryotes and organellesprokaryotes and organellesprokaryotes and organellesprokaryotes and organelles via hydrolysis (3via hydrolysis (3via hydrolysis (3via hydrolysis (3’’’’-OH)-OH)-OH)-OH) of eukaryotesof eukaryotesof eukaryotesof eukaryotes

SpliceosomeSpliceosomeSpliceosomeSpliceosome pre-mRNA splicing via pre-mRNA splicing via pre-mRNA splicing via pre-mRNA splicing via eukaryotic pre-mRNAs eukaryotic pre-mRNAs eukaryotic pre-mRNAs eukaryotic pre-mRNAs(U2 + U6 snRNAs) (U2 + U6 snRNAs) (U2 + U6 snRNAs) (U2 + U6 snRNAs) 70; 5070; 5070; 5070; 50 180; 100 ransesterification (3 180; 100 ransesterification (3 180; 100 ransesterification (3 180; 100 ransesterification (3’’’’OH)OH)OH)OH)

RibosomeRibosomeRibosomeRibosome >900 >900 >900 >900 2 2 2 2’’’’600600600600 peptidyl transfer (amide) peptidyl transfer (amide) peptidyl transfer (amide) peptidyl transfer (amide) prokaryotes and eukaryotes prokaryotes and eukaryotes prokaryotes and eukaryotes prokaryotes and eukaryotes(23S rRNA)(23S rRNA)(23S rRNA)(23S rRNA)

Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)Naturally occurring ribozymes and ribonucleoprotein enzymes (2002)

peptidyl transfer (amide)peptidyl transfer (amide)peptidyl transfer (amide)peptidyl transfer (amide)转移肽（酰胺） self-splicingself-splicingself-splicingself-splicing自我剪接 self-cleavageself-cleavageself-cleavageself-cleavage自我裂解 2222’’’’,3,3,3,3’’’’-cyclic phosphate-cyclic phosphate-cyclic phosphate-cyclic phosphate 2'2'2'2'，3' -3' -3' -3' -环磷酸 transesterificationtransesterificationtransesterificationtransesterification酯交换 prokaryotes and eukaryotesprokaryotes and eukaryotesprokaryotes and eukaryotesprokaryotes and eukaryotes 原核生物和真核生物 pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3pre-tRNA processing via hydrolysis (3’’’’-OH)-OH)-OH)-OH) 处理经水解的前氨酰- tRNA- tRNA- tRNA- tRNA（3' - OH3' - OH3' - OH3' - OH）

天然核酶及核糖酶

3.3.3.3.结构（Structure Structure Structure Structure ）As with proteins, we considerAs with proteins, we considerAs with proteins, we considerAs with proteins, we consider............

Primary:Primary:Primary:Primary: 一级

GGCCGAACUGGUAGGCCGAACUGGUAGGCCGAACUGGUAGGCCGAACUGGUA

SecondarySecondarySecondarySecondary 二级:

TertiaryTertiaryTertiaryTertiary 三级:

3.1 3.1 3.1 3.1 Primary StructurePrimary StructurePrimary StructurePrimary Structure一级结构

Limited to ribonucleotidesLimited to ribonucleotidesLimited to ribonucleotidesLimited to ribonucleotides核苷酸 ( ( ( (basebasebasebase碱基 + ribose + ribose + ribose + ribose核糖 + phosphate + phosphate + phosphate + phosphate磷酸):):):):

Common Bases:Common Bases:Common Bases:Common Bases:常见碱基

Uncommon Bases:Uncommon Bases:Uncommon Bases:Uncommon Bases:罕见碱基

N

N

N

N

O

N

N

N

N

N

N

N

N

O

N

N

N

O

O

A G C UA G C UA G C UA G C U

N

N

O

O

N

N

N

N

O

PseudouridinePseudouridinePseudouridinePseudouridine 假尿苷

Inosine Inosine Inosine Inosine 肌苷 etc... etc... etc... etc...

3.2 3.2 3.2 3.2 Secondary StructureSecondary StructureSecondary StructureSecondary Structure（二级结构）

Watson-Crick Base PairingWatson-Crick Base PairingWatson-Crick Base PairingWatson-Crick Base Pairing沃森----克里克碱基配对

Helix FormationHelix FormationHelix FormationHelix Formation螺旋形成

B-DNAB-DNAB-DNAB-DNA RNARNARNARNA

RNA usually assumesRNA usually assumesRNA usually assumesRNA usually assumesA-form helicesA-form helicesA-form helicesA-form helices…………DNADNADNADNA常常假设A-A-A-A-型螺旋

Small pore along helical axisSmall pore along helical axisSmall pore along helical axisSmall pore along helical axis沿螺旋轴形成个小孔

““““RungsRungsRungsRungs”””” stack obliquely to axis stack obliquely to axis stack obliquely to axis stack obliquely to axis 阶梯式的斜上升的中心轴

A-DNAA-DNAA-DNAA-DNA

3′

5′

-5B

V

C U GY

GG A A

-5B

V

I

10

G

AA

AC

C

UUA

AH

A

40

30

50

20

2-2 发卡型核酶二级结构

Secondary StructureSecondary StructureSecondary StructureSecondary Structure（二级结构）

Conserved base-pairing interactions result in...Conserved base-pairing interactions result in...Conserved base-pairing interactions result in...Conserved base-pairing interactions result in...保守的碱基配对相互作用的导致：

• Three Three Three Three ““““stemstemstemstem”””” regions regions regions regions 三个结构域

• Uridine-containing turnUridine-containing turnUridine-containing turnUridine-containing turn 旋转而封闭的尿苷

• An An An An ““““augmenting helixaugmenting helixaugmenting helixaugmenting helix”””” joining stems II and III joining stems II and III joining stems II and III joining stems II and III

结构域II II II II 与 III III III III连接形成的增强螺旋结构

RibozymeRibozymeRibozymeRibozyme vs. vs. vs. vs. tRNAtRNAtRNAtRNAPhePhePhePhe 核酶 苯丙氨酸tRNA tRNA tRNA tRNA

foldingfoldingfoldingfolding折叠

The hammerhead ribozyme (The hammerhead ribozyme (The hammerhead ribozyme (The hammerhead ribozyme (锤头状核酶))))

- discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986)- discovered in small RNA satellites of small viruses (1986) 是小病毒携带的小的RNARNARNARNA- replication by rolling circle mechanism- replication by rolling circle mechanism- replication by rolling circle mechanism- replication by rolling circle mechanism 通过不断循环机制进行复制折叠

Secondary structureSecondary structureSecondary structureSecondary structure

RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP） is a is a is a is a ribozymeribozymeribozymeribozymeRNAse P cleaves the 5RNAse P cleaves the 5RNAse P cleaves the 5RNAse P cleaves the 5’’’’ end of pre- end of pre- end of pre- end of pre-tRNAstRNAstRNAstRNAs

（核糖核酸酶PPPP切割5'5'5'5'前体tRNAtRNAtRNAtRNA的末段）

It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and It is composed of 12 kDa P protein and about 400 nt long RNAabout 400 nt long RNAabout 400 nt long RNAabout 400 nt long RNA

（它是由12 kDa12 kDa12 kDa12 kDa蛋白质和长约400400400400元的RNARNARNARNA组成）

The catalytic activity lies entirely within The catalytic activity lies entirely within The catalytic activity lies entirely within The catalytic activity lies entirely within RNA partRNA partRNA partRNA part

（催化活性中心位于RNARNARNARNA上）

RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP） is a is a is a is a ribozymeribozymeribozymeribozymeEnzyme is efficient without P protein but Enzyme is efficient without P protein but Enzyme is efficient without P protein but Enzyme is efficient without P protein but in high salt conditionsin high salt conditionsin high salt conditionsin high salt conditions

（在高的盐条件，酶在没有PPPP蛋白时还是有活性的）P protein or high salt is thought to P protein or high salt is thought to P protein or high salt is thought to P protein or high salt is thought to screen the repulsive electrostatic screen the repulsive electrostatic screen the repulsive electrostatic screen the repulsive electrostatic interactions between RNAse P RNA and interactions between RNAse P RNA and interactions between RNAse P RNA and interactions between RNAse P RNA and substrate pre-tRNAsubstrate pre-tRNAsubstrate pre-tRNAsubstrate pre-tRNA

(P (P (P (P 蛋白质或高盐被认为是核糖核酸酶PPPP RNA RNA RNA RNA 与前体tRNAtRNAtRNAtRNA表面之间静电排斥反应的屏障。

RNAse PRNAse PRNAse PRNAse P（核糖核酸酶PPPP）

3.3 3.3 3.3 3.3 tertiary structuretertiary structuretertiary structuretertiary structure（三级结构）

锤头状核酶 发卡状核酶 内含子IIII型核酶丁型肝炎病毒

Hammerhead ribozymeHammerhead ribozymeHammerhead ribozymeHammerhead ribozyme（锤头状核酶）

The hammerhead ribozyme The hammerhead ribozyme The hammerhead ribozyme The hammerhead ribozyme ((((锤头状核酶))))

- tertiary structure- tertiary structure- tertiary structure- tertiary structure

Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996Scott et al and Klug, Science 1996

The hairpin ribozyme (The hairpin ribozyme (The hairpin ribozyme (The hairpin ribozyme (发夹状核酶))))

From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)

The hepatitis delta ribozyme The hepatitis delta ribozyme The hepatitis delta ribozyme The hepatitis delta ribozyme ((((丁型肝炎核酶))))

From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)

Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA) Group I &II intron ribozyme (rRNA and mt RNA)内含子IIII型和IIIIIIII型核酶

Doudna and CechDoudna and CechDoudna and CechDoudna and CechNature, 2002Nature, 2002Nature, 2002Nature, 2002

内含子I型核酶(rRNA and mt RNA)

Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA) Group I intron ribozyme (rRNA and mt RNA)

Golden et al, and cechGolden et al, and cechGolden et al, and cechGolden et al, and cech Science (1998)Science (1998)Science (1998)Science (1998)

4.4.4.4.核酶的功能 （The The The The ActivityActivityActivityActivity of of of of Ribozym Ribozym Ribozym Ribozym ）

1. 1. 1. 1. 核苷酸转移作用。 　　2. 2. 2. 2. 水解反应，即磷酸二酯酶作用。 　　3. 3. 3. 3. 磷酸转移反应，类似磷酸转移酶作

用。 　　4. 4. 4. 4. 脱磷酸作用，即酸性磷酸酶作用。 　　5. RNA5. RNA5. RNA5. RNA内切反应，即RNARNARNARNA限制性内切酶

作用。

5.5.5.5.mechanism of catalysismechanism of catalysismechanism of catalysismechanism of catalysis催化反应机理

From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)From Lilley TIBS (2003)

How to catalyse the reaction ?How to catalyse the reaction ?How to catalyse the reaction ?How to catalyse the reaction ?催化反应如何进行？

Catalytic efficiency, conditionCatalytic efficiency, conditionCatalytic efficiency, conditionCatalytic efficiency, condition催化效率和条件

- ribozyme follows a Michaelis-Menten kinetics- ribozyme follows a Michaelis-Menten kinetics- ribozyme follows a Michaelis-Menten kinetics- ribozyme follows a Michaelis-Menten kinetics（米氏动力学）

EEEE（酶） + S + S + S + S（底物） ESESESES（过渡态） E + P E + P E + P E + P kkkk1111 kkkk2222

kkkk-1-1-1-1

- all ribozyme need cations for activity (Mg- all ribozyme need cations for activity (Mg- all ribozyme need cations for activity (Mg- all ribozyme need cations for activity (Mg2+2+2+2+ ,Mn ,Mn ,Mn ,Mn2+2+2+2+)))) 所有活化的核酶的需要阳离子(Mg2+ ,Mn2+)(Mg2+ ,Mn2+)(Mg2+ ,Mn2+)(Mg2+ ,Mn2+)

KKKKmmmm（米氏常数）= = = = kkkk-1-1-1-1++++ kkkk2 2 2 2

kkkk1111

= 10= 10= 10= 10-5-5-5-5-10-10-10-10-7-7-7-7 M M M M kkkkcatcatcatcat（催化常数）= 0.5-2 min= 0.5-2 min= 0.5-2 min= 0.5-2 min-1-1-1-1

kkkkcatcatcatcat/ K/ K/ K/ Kmmmm= 10= 10= 10= 103333-10-10-10-106 6 6 6 MMMM-1-1-1-1.min.min.min.min-1-1-1-1 Good catalytic efficiency!!Good catalytic efficiency!!Good catalytic efficiency!!Good catalytic efficiency!!良好的催化效率

mechanism of catalysismechanism of catalysismechanism of catalysismechanism of catalysis催化机理

IntactIntactIntactIntactPhosphodiesterPhosphodiesterPhosphodiesterPhosphodiester完整的磷酸二酯 MgMgMgMg2+2+2+2+CoordinatedCoordinatedCoordinatedCoordinated

TransitionTransitionTransitionTransitionMg2 +Mg2 +Mg2 +Mg2 +协调的过渡态

CleavedCleavedCleavedCleavedPhosphodiesterPhosphodiesterPhosphodiesterPhosphodiester结合后的磷酸二酯

Catalytic MechanismCatalytic MechanismCatalytic MechanismCatalytic Mechanism（催化机理）New crystal structure shows five MgNew crystal structure shows five MgNew crystal structure shows five MgNew crystal structure shows five Mg2+2+2+2+ sites: sites: sites: sites:新发现的结晶结构是五个 Mg Mg Mg Mg2+2+2+2+的结合域

�

核酶的剪接机制

鸟苷的3’-OH攻击内含子的5’剪接点，并与Intron的5’-端磷酸基形成共价键连接，同时5’外显子脱落下来，但3’外显子此时仍与Intron相连。5’-端外显子的3’-OH进攻内含子的3’剪接点并使413nt的内含子脱落，同时5’外显子与3’外显子连接成成熟rRNA

3D structure of ribozyme: mechanism of catalysis3D structure of ribozyme: mechanism of catalysis3D structure of ribozyme: mechanism of catalysis3D structure of ribozyme: mechanism of catalysishairpin ribozymehairpin ribozymehairpin ribozymehairpin ribozyme（发夹状核酶）

Hepatitis delta ribozymeHepatitis delta ribozymeHepatitis delta ribozymeHepatitis delta ribozyme（丁型肝炎核酶）

Ruppert et al, Nature 2001, Science 2002Ruppert et al, Nature 2001, Science 2002Ruppert et al, Nature 2001, Science 2002Ruppert et al, Nature 2001, Science 2002 Ferre dFerre dFerre dFerre d’’’’Amare, Nature 1998Amare, Nature 1998Amare, Nature 1998Amare, Nature 1998

Structure of the hairpin ribozymeStructure of the hairpin ribozymeStructure of the hairpin ribozymeStructure of the hairpin ribozyme((((发夹状核酶的结构))))

hairpin ribozymehairpin ribozymehairpin ribozymehairpin ribozyme

Ruppert et al, Nature 2001, Science 2002

hairpin ribozymehairpin ribozymehairpin ribozymehairpin ribozyme((((发夹状核酶))))

Ruppert et al, Nature 2001 Ruppert et al, Science 2002Ruppert et al, Nature 2001 Ruppert et al, Science 2002Ruppert et al, Nature 2001 Ruppert et al, Science 2002Ruppert et al, Nature 2001 Ruppert et al, Science 2002

Transition stateTransition stateTransition stateTransition state（过渡态）Ground stateGround stateGround stateGround state（基态）

hairpin ribozymehairpin ribozymehairpin ribozymehairpin ribozyme(发夹状核酶)

Ruppert et al, Science 2002Ruppert et al, Science 2002Ruppert et al, Science 2002Ruppert et al, Science 2002

Transition stateTransition stateTransition stateTransition state((((过渡态))))

5.5.5.5.RibozymeRibozymeRibozymeRibozyme应用

RibozymeRibozymeRibozymeRibozyme的发现使我们认识到酶不一定都是蛋白质，这些Ri-bozymeRi-bozymeRi-bozymeRi-bozyme主要用在RNARNARNARNA分子的切割与降解上。因为真核生物的突变体很难获得，假如用特制的RibozymeRibozymeRibozymeRibozyme破坏切割特定的RNARNARNARNA分子，使这种RNARNARNARNA不能表达出相应的蛋白质，我们再进一步观察其表型变化，从而了解这个基因的功能。另外在医学方面，可以用特别的RibozymeRibozymeRibozymeRibozyme切割一些对人体有害的RNARNARNARNA，达到解除有害基因功能的目的，这也是基因治疗的又一条路子。在动植物育种方面，可以将具有破坏病毒RNARNARNARNA的RibozymeRibozymeRibozymeRibozyme基因导入动植物细胞，提高动植物抗病毒能力。

举例：Engineered allosteric ribozymes as biosensorsEngineered allosteric ribozymes as biosensorsEngineered allosteric ribozymes as biosensorsEngineered allosteric ribozymes as biosensors（具有生物传感器功能的变构核酶）

a. a. a. a. ATP-sensing aptamer + self-cleaving ATP-sensing aptamer + self-cleaving ATP-sensing aptamer + self-cleaving ATP-sensing aptamer + self-cleaving ribozymeribozymeribozymeribozyme.... ATPATPATPATP传感适体++++自我切割核酶。

b. b. b. b. This variant favors ADP binding 100-fold This variant favors ADP binding 100-fold This variant favors ADP binding 100-fold This variant favors ADP binding 100-fold overATPoverATPoverATPoverATP. . . . 这种变异使对ATPATPATPATP的亲和力是ADPADPADPADP的100100100100倍。

c. c. c. c. Can be used as an in vitro sensor forCan be used as an in vitro sensor forCan be used as an in vitro sensor forCan be used as an in vitro sensor for enzymeenzymeenzymeenzyme activity (or for enzyme inhibition)activity (or for enzyme inhibition)activity (or for enzyme inhibition)activity (or for enzyme inhibition).... 用作体外传感器的酶激动剂（或酶的抑制剂）。

蓝箭头显示裂解位点

Most common output is Green Fluorescent Most common output is Green Fluorescent Most common output is Green Fluorescent Most common output is Green Fluorescent Protein (GFP)Protein (GFP)Protein (GFP)Protein (GFP)

最常见的输出是绿色荧光蛋白（GFPGFPGFPGFP）