Functional nucleic acidsFunctional nucleic acidsFunctional nucleic acids Functional nucleic acids from Mother Nature and from Mother Nature and by in vitro evolutionby in vitro evolution

Gust Lecture 1 byGust Lecture 1 by

DrDr YingfuYingfu LiLiDr. Dr. Yingfu Yingfu LiLi

January 6, 2010January 6, 2010

Key Historic EventsKey Historic Eventsyy

1869: DNA 1869: DNA discovered by F. discovered by F. MeischerMeischer19101910: Genes on chromosomes; T: Genes on chromosomes; T. H. H. . MorganMorgan1941: One gene1941: One gene--one enzyme, Beadle & one enzyme, Beadle & TatumTatum1944: DNA is genetic material; Avery, 1944: DNA is genetic material; Avery, McleodMcleod& & McCartyMcCarty1953 Structure of DNA Watson Crick Franklin 1953 Structure of DNA Watson Crick Franklin WilkinsWilkins1953: Structure of DNA; Watson, Crick, Franklin, 1953: Structure of DNA; Watson, Crick, Franklin, WilkinsWilkins1961: Discovery of mRNA; Brenner, Jacob & 1961: Discovery of mRNA; Brenner, Jacob & MeselesonMeseleson1966: Finished unraveling the code; Nirenberg & 1966: Finished unraveling the code; Nirenberg & KhoranaKhorana1972: Recombinant DNA made in vitro; P 1972: Recombinant DNA made in vitro; P BergBerg1972: Recombinant DNA made in vitro; P. 1972: Recombinant DNA made in vitro; P. BergBerg1973: DNA cloned on a plasmid; H. Boyer & S. 1973: DNA cloned on a plasmid; H. Boyer & S. CohenCohen1973: Discovery of reverse transcriptase; H. 1973: Discovery of reverse transcriptase; H. TeminTemin1977: 1977: DNA DNA sequencing; F. Sanger & W. sequencing; F. Sanger & W. GilbertGilbertq g; gq g; g1977: Discovery of split genes; 1977: Discovery of split genes; Sharp & RobertsSharp & Roberts1982: Discovery of ribozymes; T. 1982: Discovery of ribozymes; T. CechCech & S. & S. AltmanAltman1986: Creation of PCR; K. 1986: Creation of PCR; K. MullisMullis1990: Invention of in vitro selection; J. Szostak, L. Gold and G. Joyce1990: Invention of in vitro selection; J. Szostak, L. Gold and G. Joyce2002: Discovery of 2002: Discovery of riboswitches; R. Breakerriboswitches; R. Breaker

Discovery of RibozymesDiscovery of Ribozymesy yy y

Nobel prize in 1989Nobel prize in 1989Nobel prize in 1989Nobel prize in 1989

Sidney AltmanSidney Altman

Tom Tom CechCech

Group I Group I IntronIntron Ribozyme: Catalyzing Ribozyme: Catalyzing selfself--splicing of certain splicing of certain intronsintrons (1982)(1982)

RNase P RNase P RNA: the catalytic component RNA: the catalytic component for for tRNAtRNA processing (1983)processing (1983)

Doudna & Cech 2002. The chemical repertoire of natural ribozymes. Nature 418:222–28

‘RNA World’ Hypothesis‘RNA World’ Hypothesisypyp

‘‘RNA world’ there was a stage early in the early RNA world’ there was a stage early in the early ‘‘RNA world’: there was a stage early in the early RNA world’: there was a stage early in the early evolution of life that was dominated by RNA.evolution of life that was dominated by RNA.

Moreover, the functions of RNA in modern cells are Moreover, the functions of RNA in modern cells are only remnants of its previous roles.only remnants of its previous roles.

Walter Gilbert, Walter Gilbert, 1986198619861986

Remnants Remnants of the of the ‘RNA World’?‘RNA World’?

11 SelfSelf--splicing splicing intronsintrons (group I and group II)(group I and group II)1.1. SelfSelf--splicing splicing intronsintrons (group I and group II)(group I and group II)

2.2. RNase P RNase P (ribozyme that cleaves (ribozyme that cleaves tRNAtRNA precursors)precursors)

SelfSelf cleaving viral RNAs cleaving viral RNAs (h h d h i i (h h d h i i 3.3. SelfSelf--cleaving viral RNAs cleaving viral RNAs (hammerhead, hairpin, (hammerhead, hairpin, HDV and VS ribozymes)HDV and VS ribozymes)

44 PeptidylPeptidyl transferasetransferase in the ribosomein the ribosome4.4. PeptidylPeptidyl transferasetransferase in the ribosomein the ribosome

5.5. RibonRibonucleotidesucleotides involved in: involved in: metabolism (e g ATP UTP NADH NADPH)metabolism (e g ATP UTP NADH NADPH) metabolism (e.g., ATP, UTP, NADH, NADPH)metabolism (e.g., ATP, UTP, NADH, NADPH)

signaling (signaling (cAMPcAMP, , cGMPcGMP, GTP, ITP), GTP, ITP)

assembly of complexes (GTP and ATP)assembly of complexes (GTP and ATP)y p ( )y p ( )

eenergynergy for motility, ion pumping, etc. (ATP, GTP)for motility, ion pumping, etc. (ATP, GTP)

Invention of SELEXInvention of SELEX

Jack SzostakJack Szostak

Larry GoldLarry GoldLarry Go dLarry Go d

Gerald JoyceGerald Joyce: :

In In vitro selection (SELEX)vitro selection (SELEX)

Discovery of RiboswitchesDiscovery of Riboswitchesyy

Ron Ron Breaker, 2002Breaker, 2002

1)1) Many riboswitch are now Many riboswitch are now knoenknoen, each responds , each responds to to a specific metabolite (such as a specific metabolite (such as an amino acid or coenzyme). an amino acid or coenzyme).

2)2) Each riboswitch contains a receptor domain (aptamer) and expression platformEach riboswitch contains a receptor domain (aptamer) and expression platform3)3) A A riboswitch riboswitch can repress can repress gene expression gene expression by by controlling (a) transcription controlling (a) transcription

termination and (b) translation termination and (b) translation initiation.initiation.

Riboswitch Aptamer StructuresRiboswitch Aptamer Structurespp

Riboswitch aptamers for Riboswitch aptamers for ((a) a) purinepurine, , (b(b) ) TPP TPP (b(b) ) TPP, TPP, ((c) c) SS--adenosylmethionineadenosylmethionine

(SAM), class(SAM), class--I, I, ((dd) SAM, class) SAM, class--II, II, (( ))(e) SAM class(e) SAM class--III, III, (f(f) ) lysine, lysine, (g) (g) GlcN6PGlcN6P, , (h) (h) MgMg22++(h) (h) MgMg22++

Classes of Functional Nucleic AcidsClasses of Functional Nucleic Acids

1.1. Aptamers (both Aptamers (both DNA and RNA DNA and RNA based)based)))

2.2. Nucleic acid enzymes Nucleic acid enzymes (ribozymes (ribozymes (ribozymes, (ribozymes, DNAzymes and DNAzymes and aptazymes)aptazymes)

3.3. Riboswitches Riboswitches (aptamer linked to (aptamer linked to e p ession platfo me p ession platfo mexpression platformexpression platform

Navani & Li, Curr Navani & Li, Curr Opin Chem BiolOpin Chem Biol 20062006

Aptamers for Small MoleculesAptamers for Small Moleculespp

Lau and Lau and Li, in pressLi, in press

Aptamers for Small MoleculesAptamers for Small Moleculespp

Small-molecule target Kd, µM Reference ethanolamine 0.006 * 126 Small-molecule target Kd, µM Reference ethanolamine 0.006 * 126

Small-molecule target Kd, µM Reference tobramycin 0.002 160,161 sialyl Lewis X (sLeX) 0.003 174 cyanocobalamin (vitamin B12) 0 09 108

Small-molecule target Kd, µM Reference tobramycin 0.002 160,161 sialyl Lewis X (sLeX) 0.003 174 cyanocobalamin (vitamin B12) 0 09 108 ethanolamine 0.006

-estradiol 0.1 274 N-methylmesoporphyrin IX (NMM) 0.4 32 cocaine 0.4 271 cellobiose 0.6 37 sulforhodamine B dye 0 7 36

ethanolamine 0.006 -estradiol 0.1 274 N-methylmesoporphyrin IX (NMM) 0.4 32 cocaine 0.4 271 cellobiose 0.6 37 sulforhodamine B dye 0 7 36

cyanocobalamin (vitamin B12) 0.09 theophylline 0.1 90 S-adenosylhomocysteine 0.1 97 neomycin 0.1 105 kanamycin B 0.2 164 L-arginine 0.3 92 sulforhodamine B dye 0.3 112 8-oxo-deoxyguanosine 0.3 95 fl i l id (FMN) 0 5 99

cyanocobalamin (vitamin B12) 0.09 theophylline 0.1 90 S-adenosylhomocysteine 0.1 97 neomycin 0.1 105 kanamycin B 0.2 164 L-arginine 0.3 92 sulforhodamine B dye 0.3 112 8-oxo-deoxyguanosine 0.3 95 fl i l id (FMN) 0 5 99 sulforhodamine B dye 0.7

(R)-thalidomide 1 276 cholic acid 5 40 adenosine / ATP 6 102 GTP 10 39 reactive green 19 dye 30 26

sulforhodamine B dye 0.7 (R)-thalidomide 1 276 cholic acid 5 40 adenosine / ATP 6 102 GTP 10 39 reactive green 19 dye 30 26

flavin mononucleotide (FMN) 0.5 99 7-methyl-guanosine 0.5 93 coenzyme A (CoA) 0.5 94 4,4’-methylenedianiline 0.5 98 ATP 0.7 24 riboflavin 1 91 guanine 1 103 streptomycin 1 96

flavin mononucleotide (FMN) 0.5 99 7-methyl-guanosine 0.5 93 coenzyme A (CoA) 0.5 94 4,4’-methylenedianiline 0.5 98 ATP 0.7 24 riboflavin 1 91 guanine 1 103 streptomycin 1 96

RNA RNA

DNA aptamersDNA aptamers

reactive green 19 dye 30 L-tyrosinamide 50 42 L-arginine 3000 39

reactive green 19 dye 30 L-tyrosinamide 50 42 L-arginine 3000 39

Malachite Green dye 1 158 tetracycline 1 165 chloramphenicol 2 110 L-dopamine 2 111 nicotinamide mononucleotide (NMN) 3 91 codeine 3 107 ATP (recognizing triphosphate) 5 101 biotin 6 113

Malachite Green dye 1 158 tetracycline 1 165 chloramphenicol 2 110 L-dopamine 2 111 nicotinamide mononucleotide (NMN) 3 91 codeine 3 107 ATP (recognizing triphosphate) 5 101 biotin 6 113

aptamersaptamers

biotin 6 viomycin 10 166 cAMP 10 106 adenine 10 104 D-tryptophan 20 25 L-tyrosine 40 172 flavin adenine dinucleotide (FAD) 50 167 L-arginine 60 171 L citrulline 60 171

biotin 6 viomycin 10 166 cAMP 10 106 adenine 10 104 D-tryptophan 20 25 L-tyrosine 40 172 flavin adenine dinucleotide (FAD) 50 167 L-arginine 60 171 L citrulline 60 171

Book chapter by Scott Book chapter by Scott Siverman, 2009Siverman, 2009

L-citrulline 60 171 Cibacron Blue 3G-A dye 100 1 L-arginine 200 170 L-isoleucine 200 173 Reactive Blue 4 dye 600 1 L-isoleucine 1000 17 L-arginine 4000 20 L-valine 10000 19

L-citrulline 60 171 Cibacron Blue 3G-A dye 100 1 L-arginine 200 170 L-isoleucine 200 173 Reactive Blue 4 dye 600 1 L-isoleucine 1000 17 L-arginine 4000 20 L-valine 10000 19

Aptamers for ProteinsAptamers for Proteinspp

Protein target Kd, nM Reference 43

Protein target Kd, nM Reference 43

Protein target Kd, nM Reference keratinocyte growth factor 0.0003 177 vascular endothelial growth factor (VEGF) 0.05 59 thrombin 0.05 68 HIV 1 T t t i 0 1 30

Protein target Kd, nM Reference keratinocyte growth factor 0.0003 177 vascular endothelial growth factor (VEGF) 0.05 59 thrombin 0.05 68 HIV 1 T t t i 0 1 30 platelet-derived growth factor B-chain 0.1 43

MUC1 tumor marker 0.1 22 L-selectin 0.3 45 BcsI 10 34 immunoglobulin IgE 10 44

platelet-derived growth factor B-chain 0.1 43 MUC1 tumor marker 0.1 22 L-selectin 0.3 45 BcsI 10 34 immunoglobulin IgE 10 44

HIV-1 Tat protein 0.1 30 P-selectin 0.2 178 haemagglutinin 0.2 76 basic fibroblast growth factor 0.4 53 human CD4 0.4 78 rat GPCR for neurotensin 0.4 123 coagulation factor IXa 0.6 69 nuclear factor B (NF-B) 1 62 antibody against human insulin receptor 2 80 interferon- 2 57

HIV-1 Tat protein 0.1 30 P-selectin 0.2 178 haemagglutinin 0.2 76 basic fibroblast growth factor 0.4 53 human CD4 0.4 78 rat GPCR for neurotensin 0.4 123 coagulation factor IXa 0.6 69 nuclear factor B (NF-B) 1 62 antibody against human insulin receptor 2 80 interferon- 2 57 immunoglobulin IgE 10

thrombin 30 38 anti-MUC1 IgG3 monoclonal antibody 50 21 ricin 60 140 tenascin-C 150 47

immunoglobulin IgE 10 thrombin 30 38 anti-MUC1 IgG3 monoclonal antibody 50 21 ricin 60 140 tenascin-C 150 47

human nonpancreatic secretory phospholipase A2 2 60 human complement C5 component 2 63 G6-9 anti-DNA autoantibody 2 179 colicin E3 2 75 transforming growth factor- type III receptor 2 180 elongation factor Tu (EF-Tu) 3 54 L-selectin 3 83 formamidopyrimidine glycosylase (Fpg) 3 70 angiopoietin-2 3 72 HIV 1 reverse transcriptase (RT) 5 49

human nonpancreatic secretory phospholipase A2 2 60 human complement C5 component 2 63 G6-9 anti-DNA autoantibody 2 179 colicin E3 2 75 transforming growth factor- type III receptor 2 180 elongation factor Tu (EF-Tu) 3 54 L-selectin 3 83 formamidopyrimidine glycosylase (Fpg) 3 70 angiopoietin-2 3 72 HIV 1 reverse transcriptase (RT) 5 49

RNA RNA

DNA aptamersDNA aptamers

misactivated tRNA synthetase 15000 23 misactivated tRNA synthetase 15000 23 HIV-1 reverse transcriptase (RT) 5 bacteriophage R17 coat protein 5 50 extracellular regulated kinase 2 (ERK2) 5 31 fibrinogen-like domain of tenascin-C 5 88 HIV-1 surface glycoprotein gp120 5 134 hepatitis C virus nonstructural protein 3 protease 6 65 antibody against MIR of AChR 6 117 streptavidin 7 116 coagulation factor VIIa 10 181 cytotoxic T cell antigen-4 (CTLA-4) 10 73

58

HIV-1 reverse transcriptase (RT) 5 bacteriophage R17 coat protein 5 50 extracellular regulated kinase 2 (ERK2) 5 31 fibrinogen-like domain of tenascin-C 5 88 HIV-1 surface glycoprotein gp120 5 134 hepatitis C virus nonstructural protein 3 protease 6 65 antibody against MIR of AChR 6 117 streptavidin 7 116 coagulation factor VIIa 10 181 cytotoxic T cell antigen-4 (CTLA-4) 10 73

58

aptamersaptamers

elongation factor SelB 20 58 pepocin 20 67 prion protein (PrPSc) 20 182 immunoglobulin IgE 30 44 HTLV-1 Rex ARM peptide 30 61 Alzheimer’s A4(1-40) amyloid peptide 30 183 heparan sulfate (on cell surface) 40 184 human epidermal growth factor receptor-3 50 74 herpes simplex virus-1 US11 protein 70 146 HIV-1 integrase 80 55

elongation factor SelB 20 58 pepocin 20 67 prion protein (PrPSc) 20 182 immunoglobulin IgE 30 44 HTLV-1 Rex ARM peptide 30 61 Alzheimer’s A4(1-40) amyloid peptide 30 183 heparan sulfate (on cell surface) 40 184 human epidermal growth factor receptor-3 50 74 herpes simplex virus-1 US11 protein 70 146 HIV-1 integrase 80 55 HIV 1 integrase 80 prion protein (PrP) 100 86 fibronectin (on cell surface) 100 184 substance P 200 87 human nerve growth factor 200 56 streptavidin 200 85 laminin (on cell surface) 200 184 Ras-binding domain of Raf-1 200 71 HIV-1 Tat protein 300 66 thrombospondin (on cell surface) 400 184

HIV 1 integrase 80 prion protein (PrP) 100 86 fibronectin (on cell surface) 100 184 substance P 200 87 human nerve growth factor 200 56 streptavidin 200 85 laminin (on cell surface) 200 184 Ras-binding domain of Raf-1 200 71 HIV-1 Tat protein 300 66 thrombospondin (on cell surface) 400 184

Book chapter by Scott Book chapter by Scott Siverman, 2009Siverman, 2009

Diverse RiboswitchesDiverse Riboswitches

Functional Nucleic Acid TodayFunctional Nucleic Acid Todayyy

1.1. Thousands of original papers and reviews on aptamersThousands of original papers and reviews on aptamersg p p pg p p p2.2. Three books on aptamers (below); more to comeThree books on aptamers (below); more to come3.3. One aptamer drug (One aptamer drug (MacugenMacugen; approved in 2004); ARC1779 ; approved in 2004); ARC1779

(from (from ArchemixArchemix) in phase II clinic trial) in phase II clinic trial(from (from ArchemixArchemix) in phase II clinic trial) in phase II clinic trial4.4. Several companies in the world (Several companies in the world (AchemixAchemix, , SomaLogicSomaLogic and and

NoxxonNoxxon AG are the three major players)AG are the three major players)