>> SO IT IS MY GREAT PLEASURE TO INTRODUCE TODAY'S NIH DIRECTORS SEMINAR SPEAKER, ADRIAN FERRE-D' AMARE. HE WAS BORN IN JAPAN BUT GREW KNOP MEXICO AND HAS A B. S. IN CHEMISTRY FROM A TECHNICAL UNIVERSITY NAMONTEREY BEFORE HE CAME TO THE UNITED STATES WHERE HE DID HIS DOCTORAL WORK AT ROCKEFELLER AND THEN HIS POST DOC AT YALE WHERE HIS [INDISCERNIBLE]. HE WAS ALSO THE RECIP CREPT CREPT--RECIPIENT OF A NUMBER OF DIFFERENT HONORS IN TERMS OF RESEARCH SUPPORT AND MOST RECENTLY, HAS BEEN AT THE FRED HUTCHISON RESEARCH CENTER AS AN HHMI SCHOLAR OR RESEARCHER. HE WAS RECRUITED HERE A COUPLE YEARS AGO AND BECAME A SENIOR INVESTIGATOR IN NHLBI WHERE HE IS CURRENTLY THE CHIEF OF THE LABORATORY OF RNA BIOPHYSICS AND CELLULAR PHYSIOLOGY. HIS WORK HAS FOCUSED ON RNA, A LOT OF DIFFERENT ASPECTS OF THE BIOLOGY AND STRUCTURE OF RNA, AND JUDGING BY THE PEOPLE WHO ARE IN THIS AUDIENCE, HE BRINGS TOGETHER AN INTERESTING ARRAY OF NIH SCIENTISTS, INTERESTED IN BIOCHEMISTRY AND GENETICS AND BIOLOGY AND I DIDN'T BRING THE TITLE OF HIS TALK WITH ME AND HE SAID SOMETHING ABOUT RNA. SO IF YOU'RE HERE TO HEAR ABOUT RNA, YOU'LL BE DELIGHTED. [LAUGHTER] ADRIAN? >> THANKS FOR THE INTRODUCTION AND THE OPPORTUNITY TO TELL YOU ALL ABOUT LEARNING THIS SINCE MOVING HERE ABOUT ASPECTS OF RNA. SO THAT'S THE TITLE, THAT'S AS MIKE POINTED OUT, WHAT IT SAYS SPECIFICALLY BUT I SHOULD PROBABLY START GIVING THE AUDIENCE BY ASKING THIS QUESTION OF WHY BOTHER STUDYING RNA. AND I THINK THERE ARE THREE ANSWERS THAT I CAN DEFEND. ONE IS THAT RNA IS IMPORTANT FOR MULTIPLE ASPECTS OF BIOLOGY, IT'S PURELY CENTRAL IN THE FLOW OF BIOLOGICAL INFORMATION, AS MESSENGER RNA, AND KORT SWRAL AND CO AUTHORS, IT SPLICES, IT INSISTS ON [INDISCERNIBLE] OF THE CELL SPLICES INTRONS AND THE LATTER, IT IS PART OF SEVERAL GENETIC ELEMENTS OF QUITE A BIT OF IMPORTANCE AND THAT BRINGS US TOCCATA LYTIC RNA, CAT OLDER PEOPLE SIS, AND FACTORS THAT ARE FAIRLY WIDE SPREAD IN BIOLOGY AND OTHERS. AND OF COURSE, THERE ARE MANY GENETIC REGULATORS MADE OF RNA, THINGS LIKE SMALL RNAs, [INDISCERNIBLE] I WILL TELL YOU MORE ABOUT THAT IN THIS A MINUTE AND MANY ENCODING RNAs LARGE AND SMALL WHICH ARE DISCOVERED EVERY DAY. SO THAT'S ONE ASPECT. THE SECOND, I GUESS THIS IS APPROPRIATE FOR THE LOCATION TODAY, IS THAT IF YOU WILL, THE OTHER BIOPOLYMER AND WHAT I MEAN BY THIS AND THE NOTION THAT THEY WORKED OUT, IN BUILDING THREE NEXT DOOR HERE IN THE 60S THAT PROTEINS FORM IN A SPECIFIC WAY WHICH IS DEFINED BY THE SEQUENCE, THE PRIMARY STRUCTURE. AND RNA, MANY RNAs INFACT HAVE THE SAME PROPERTY THAT, IS THEY FOLD INTO DEFINED, IF YOU WILL GLOBEULAR FAULT WHICH IS PURE DEFINED UNDER PHYSIOLOGICAL CONDITIONS BY THE SEQUENCE AND THAT'S REMARKABLE ON THE CLINICAL PERSPECTIVE BECAUSE THESE ARE VERY DIFFERENT BIOPOLARIZEDDENERS SO PROTEIN BACKBONE HAS 2-DEGREES OF FREEDOM AND [INDISCERNIBLE] AND IT'S ELECTRICAL WRASSE THE RNA BACKBONE CARS A CHARGE, OF RESIDUE, SO THAT'S VERY DIFFERENT AND THE SIDE CHAIN OF COURSE IS DIFFERENT AND 21, 22 DEPENDING ON HOW YOU COUNT DIFFERENT SIDE CHAINS WHICH GO ALL THE WAY, AND RESIDUES TO THE THINGS WHICH ARE ACIDIC AND CHARGED, POSTURALLY CHARGED AND IMPORTANTLY AT LEAST TWO CHAINS WHICH IOHINIZE THE Ph WHICH ARE GOOD BASED CATALYST AS A CONSEQUENCE SO THIS IS FOUR NUCLEOTIDES AND BASIS AND WHICH ARE ECENTSIALLY VERY SIMILAR AND NONE OF WHICH IOINIZE THE Ph. AND DESPITE THIS RNA CAN FOLD IN SPECIFIC MANNER AND SO THAT'S QUITE REMARKABLE AND I WOULD POSE THAT BY STUDYING RNA CARRIES OUT WHAT PART STATES IN THINGS LIKE FOLDING, RECOGNITION OF SMALL MOLECULES, CAT TOLL-LIKE RECEPTOR SIS AND COMPARING THOSE KNOWLEDGE WITH WHAT WE'VE LEARNED OVER THE COURSE OF THE LAST 300 YEAR FROM PROPROPANE TO THE SAME THING, WE MIGHT BE ABLE TO DISENTANGLE SOME OF THE UNDERLYING PRINCIPLES WHICH ALLOW A POLYMER MAYBE TO CARRY OUT SUCH FUNCTIONS. AND LASTLY, BUT NOT LEAST, IS THE FACT THAT RNA IS BOTH PROVEN AND THE POTENTIAL TARGET FOR THE INTERIVATION, SO SOMETHING LIKE 85% OF ANTIBIOTICS WHICH ARE CURRENTLY IN CLINICAL USE TARGET THE RIBOSOME AND NOT JUST THE RIBOSOME IN GENERAL, BUT THE RNA, BUT IN THAT SENSE RNA IS PROVEN DRUG TARGET BUT CAN YOU ARGUE RIBOSOME HAS LARGE RNAASKS IN FACT IT'S A FRYERY WITH AND IT HAS MOLECULAR DIVERSITY AND COMPLEXITY TO ACHIEVE TO ALLOW FOR SPECIFIC TARGETING BY SMALL MOLECULES, THAT'S SOMETHING I WILL DISCUSS IN A FEW MINUTES JUST LIKE MANY GROUPS OF INTERESTING TARGETING PROPANES FROM ACTIVATING ENZYMES TO TARGETING THINGS LIKE [INDISCERNIBLE] AND PROTEIN-PROTEIN INTERFACES AND THEN RNA ONE CAN TARGET SOMETHING OTHER THAN THE ACTIVE SITE AND IN THINKING ABOUT DRUG DISCOVER AND DEVELOPMENT, THERE'S ALWAYS THE NEXT STEP AND THAT IS THE EVOLUTION OF DRUG THAT'S RESISTANCE AND SO, ONE WOULD LIKE TO KNOW HOW RNA EVOLVES IN RESPONSE TO SELECTIVE PRESSURES. SO, FOR THAT BACKGROUND AND TO TELL YOU IN A CONDENSED FORM ARE, THREE SHORT STORIES WHICH ADDRESS SOME OF THESE POINTS. I'LL TELL YOU FIRST ABOUT GENE REG LATTER RNA WHICH IS WIDE SPREAD BACTERIA AND EXTENDING FUNGIA AND PLANT THIS IS IS A SWITCH THAT RESPONDS TO THE ACTIVATOR B-1, THYMINE PHOSPHATE RIBO-SWITCH AND WOOL TELL YOU ABOUT A PLATFORM FOR WHAT WE USE FOR DRUG DISCOVERY. I'LL TELL YOU ABOUT RIBOSWITCH WHICH RECOMMENDS THIS MOLECULE, CYCLIC GNP AND IT'S AN IMPORTANT SECOND MESSENGER IN BACTERIAIARY AND SOMETHING MORE STRUCTURED BUT ALSO ABOUT FOLDING AND HOW IT'S ORGANIZATION MAY GIVE US SOME LINTS AS TO HOW TO TARGET IT IN A WAY OTHER THAN TO THE ADAPTIVE SITE AND LASTLY I'LL TELL YOU ABOUT THE FOREST IN THE RNA EVOLUTION AND HOW WE'RE COMBINING NEXT GENREQUENCING AND SELECTION TO LEARN SOMETHING ABOUT, IF YOU WILL THE FITNESS LANDSCAPE, THE LANDSCAPING WHICH RNA BOTH AND WHAT THAT MAY TELL US ABOUT A LOT OF OTHER THINGS, THE EVOLUTION OF DRUG RESISTANCE. SO THIS IS A--A VERY POPULAR SLIDE THAT I'LL SHOW, AND NONETHELESS, THIS IS THE ORIGIN CONCEPTUALLY OF RIBOSOME SWITCHES, SO THIS FIGURE IS TAKEN FROM CLASSIC PAPER, [INDISCERNIBLE], 1961 AND THIS PAPER, IS [INDISCERNIBLE] INTRODUCED THE NOTION OF THE REFREESOR, THE NORMALIZED REEXPRESSOR ASK THEY SAY HERE, WHICH BINDS THE LACTOSE, THAT THIS METRICS TAB O LIESED RESPONSIVE MOLECULE COULD BE PROPANE AND THEY SAY MAYBE RNA WHICH IS PRODUCED FROM THE REGULATOR GREENE AND GOES THROUGH THINGS ON THE OITERATOR. AND--OPERATOR ANDY WON THAT THE REPRESSOR TURN OUT TO BE PROPANE BUT THIS BASIC CONCEPT OF REGULATION ENTRANCED BY A SMALL MOLECULE RNA TURN OUT TO BE FUNDAMENTALLY CORRECT SO IN THE EARLY YEARS OF THIS MILLENNIUM SEVERAL GROUPS AND BREAKER AND OTHERS IN FACT, DISCOVERED RIBOSWITCHS AND DESIGN FACT GENE CONTROL ELEMENTS AND AT THIS POINT, THEY HAVE A 20 DEFINITE CLASSES KNOWN, AND RESPONDING MOLECULES RANGING FROM FLUORIDE TO TINY IONS TO THINGS LIKE VITAMIN B12. AND THIS FIGURE IS TAKEN FROM A FAIRLY OLD PAPER NOW, TO [INDISCERNIBLE] FROM THE BREAKER LAB BUT I CHOSE TWO THINGS WHEN I WANT TO DRIVE HOME, ONE IS DIVERSITY AND RIBOSWITCHES AND THE OTHER IS THAT THE GIVEN GENOME, EXAMPLE IS METHIT ACETYLASE IS ONE KIND OF SWITCH SAY THIS GREEN ONE, THIS RIBOSWITCH CONTROLS MULTIPLE DISJOINT, UPPER LO SIGNIFY AND THIS CAN--LOCI AND THIS CAN COORDINATE GENE EXPRESSION THROUGHOUT THE GENOME AND THEY DO SO BY RESPONDING TO THE SMALL MOLECULES AND IN THAT SENSE THEY'RE SIMILAR TRANSCRIPTION FACTORS WHICH CAN FUNCTION ENTRANCED AND THE DIFFERENCE IS THAT RIBOSWITCHES NOT ONLY CONTROL SPRNS SCRIPGZ, THEY'RE KNOWN TO DIFFERENT CASES CONTROL TRANSLATION, SLICING MRNA STABILITY. AND SO EACH CASE WILL BE CERTAINLY OF COURSE DIFFERENT, BUT FOR GENERAL AUDIENCE I'D LIKE TO SHOW A CARTOON FORM OF HOW TRANSCRIPTIONAL [INDISCERNIBLE] WORKS. THIS IS IDEALIZED RIBOSWITCH AND THIS IS TRANSLATED REGIONI TR OF THE GENE. IN THIS CONTEXT, IN THIS ANYWAY, THIS WILL BE MRNA WHICH IS TRANSCRIBEDDED NORMALLY WHICH IS ON IF YOU WILL AND THIS ELEMENT WHICH WILL BE THE RIBOSWITCH AND WHEN A METABOLITE, A MESSENGER ACCUMULATES, IT BINDS TO THE ELEMENT, AND BINDING ENERGY USED TO STABILIZE A STRUCTURE OF THE RNA, AND THIS PARTICULAR CARTOON, WHAT IS HAPPENING, YOU NOTE THIS IS IN ADDITION TO STABLING ELEMENT, BOUND TO METABOLITE, THIS LOOP, MELTS AND ONE OF THE STRANDS OF THAT STEM LOOP, IS THE LEFT SCREEN STRENGTH COMES TO FORM PART OF THE SMALL MOLECULE BOUND RNA. AND THAT ALLOWS IN THIS CASE, THE OTHER STRAND TO BASE WITH THE RED PORTION HERE AND WHAT THIS S&P A STEM LOOP FOLLOWED ONE OF USE WHICH IS ICONICAL INDEPENDENT [INDISCERNIBLE] SO IN THIS FORM, TRANSCRIPTION IS OFF AND THE GENE IS OFF. OKAY? SCHETHIS IS A GENERAL EXAMPLE, BUT INTRODUCES NOTION OF TWO CONFIRMATIONS AND I WON'T DISCUSS THIS IN DETAIL TODAY, CAN YOU IMAGINE THERE ARE ISSUES OF KINETIC VERSES CONTROL OF THIS PROCESS, AND THE POINT IS THAT THIS FOR HISTORIC REASONS IS DOMAIN FOR SUFFICIENT FOR BINDING THE MOLECULE IS THE [INDISCERNIBLE] DOMAIN BUT FOR STUDIES WE STUDY THIS IN ISOLATION BECAUSE IN THE CONTEXT OF THE LIGAND IT'S MORE STABLE. OKAY, SO IN THAT DIRECTION, LET ME TURN TO THE TPT OR THE RIBOSWITCH. THIS RNA WHICH BINDS [INDISCERNIBLE] PHOSPHATE WHICH IS GENETICALLY CHARGED MOLECULE AND IT HAS BEEN A CHARGED PHOSPHATE AND AT THE VERY LEVEL IT'S SURPRISE TAG RNA WHICH IS CHARGED CAN DO SO BUT IT DOES WITH THE STRUCTURE NOW MANY YEARS AGO. AND WHAT WE FOUND IS THAT IT CONSISTS OF TWO STACKS, THIS STACK AND THIS STACK WHICH SANDWICH OR A BRIDGE TOGETHER BY THE TIME [INDISCERNIBLE] PHOSPHATE AND STRUCTURALLY THIS REMARKABLE BECAUSE BOTH OF THESE HELICAL STRUCKS ARE CHARGED AND THIS INTERACTION PLUS GREEN INTERACTION HERE SEEMS TO BE SUFFICIENT TO BRING THE HELIXES SIDE BY SIDE. THIS RNA CONSERVED AS I MENTIONED FROM THE BACTERIA TO PLANTS AND IF YOU LOOK AT [INDISCERNIBLE], THEY'RE IN THE WEB HERE SO THEY ENCOMPASS BINDING POCKETS NOT SURPRISINGLY AND OTHER GROUPS SAW THE STRUCTURE OF THE PLANT, THE VERSION OF THE RIBOSWITCH, CAN YOU SEE THEY'RE IMPOSED AND YOU CAN SEE THEY'RE IDENTICAL SO THAT'S FOR CONSERVATION. NOW, THERE'S THIS QUESTION, I RAISED, HOW DOES THIS NEGAATIVELY CHARGED MODEL CITIZENETY BIND, AND THIS DOES NOT BIND WITH THE NAKED PHOS PHOSPHATE, APPROXIMATE BINDS WITH THE GROUP WITH THE TWO LEVEL CAT IONS AND IT IS THROUGH THESE CAT IONS THAT THE WATER THAT BINDS THIS ELEMENT. THE OTHER SIDE OF THE VITAMIN B-1 CONTAIN THIS IS PYRAMIDIN, AND THE PERM DIN IS LIKE A BASE, SO IT MAKES A BASE PAIR WHAT IS CALLED THE SUGAR EDGE, WHAT WOULD BE ANALOGOUS TO THIS RANK. SO THAT'S NICE. AND YOU NOTICE THAT DRUG INTERACTIONS HERE WITH THE MIDDLE. AND SO WITH ONE MOLECULE SHORTER WHICH HAVE THIS AND THAT MODEL CITIZENETY WOULD BIND RNA AND IN FACT THEY DO. SO, THE IMMEDIATE PRECURIOUSOR TO THE PHOSPHATE IS [INDISCERNIBLE] TMT, AND IT TURNS OUT THAT THIS IS BOUND BY RIBOSWITCH IN ESSENTIALLY THE SAME MANNER ON THIS PHASE WHICH IS HERE AND TWO METHYL IONS, IS TWO, STILL COORDINATE TO A SINGLE PHOSPHATE. AND THE WAY THIS IS POSSIBLE IS THAT THE RNA HAVE IT FLEXIBLE AS IT SHRINKS SUBTLEY AND THE GRAY FORM IS THE PNP STRUCTURE AND THERE'S I COST, TO THAT AND THAT'S SHOWN HERE IS A 30 FOLD DISCRIMINATION BETWEEN BINDING BTP AND [INDISCERNIBLE], AND I FOUND THAT SUFFICIENT FOR BIOLOGICAL FUNCTION. SO THAT'S HOW THIS WORKS APPARENTLY. NOW I MENTION THESE ARE AND THAT'S THE BASE ESTIMATE THAD FOR THE FUNCTION IT TURNS OUT OF A COMPOUND WHICH HAS BEEN USED ANTIBACTERIALLY AND ANTIFUNGALLY FOR OVER 50 YEARS NOW AND THIS COMPOUND FOR THE FAMINE PHOSPHATE AND IT WAS DEVELOP INDEED THE 50S, I BELIEVE LAST CENTURY TO FUNCTION AS ANTAGONIST OF FAMINE EMPLOYING ENZYMES AND IT'S A [INDISCERNIBLE] IN HYDROX OLDER PEOPLE FROM HERE AND IT GETS PHOSPHORYLATED BY THE ENZYME WHICH IS PHOSPHORYLATED IN B-1. AND IN ORGANISMS LIKE US WHICH DO NOT MAKE FAMINE, THIS IS TOXIC BECAUSE IT BINDS ENZYMES THAT EMPLOY DISCIPLINARY ROSINEI PHOSPHATE BUT AS REMARKABLE AS THE TOXIC THINGS LIKE BACTERIA AND FUNGI WHICH CAN SINGICIZE AND YOU THINK THEY WOULD UPREGULATE THE THYMINE PRODUCTION TO COMBAT THIS DRUG TTURNS OUT, AND THE LAB A FEW YEARS AGO, THAT THE TOXICITY OF THE PHOSPHATE IS DERIVED WITH BINDING TO THE RIBOSWITCH IN BACTERIA AND FUNGI, AND THIS IS BASICALLY THE SAME MODE AS THYMEIN HERE WHICH IS MISREGULATION OF THE RIBOSWITCH, AND IT CAN CAUSE TOXICITY AND THESE ARE RESISTANT IN PLANT AND FUNGI AND THOSE HAVE MUTATIONS IN THIS BINDING POCKET. I LITTLE ASSESSMENT ASIDE, I MENTION TREAD IS NO ASSESSMENT ON THIS RING, BUT THIS BIND THE SLIGHTLY HERE IN THE THYMINE IS TO MOVE THIS AWAY, SO INTERESTING CORRELARY IS THIS SAY S A BIT REMINISCENT OF HOW GLEEVEC WORKS AND IT'S THE SAME INHIBITOR OF THE KINASE AND YOU MIGHT RECALL THAT WHAT GLEEVEC DOES IT BINDS TO A NONNATIVE, NONPATHWAY FORM OF HIPPO KINASE. AND HERE, IN THIS CASE, RNA IS EXPLORING A CONFIRMATION THAT IT DOES NOT NORMALLY EXPLORE BOUND TO COGNITIVE LIAISON GANNA BOUND FOR THIS OFFICIAL MOLECULE. SO THAT SHOWS IN PRINCIPLE WE CAN TARGET WITH SO CALLED DRUGS IF YOU WILL. THIS COMPOUND IS HIGHLY TOXIC TO HUMANS AND IT'S NOT VERY USEFUL. SO, IN GOING ABOUT LOOKING FOR DRUG CANDIDATES THERE ARE SEVERAL APOUCHS, ONE WILL BE STRUCTURE GAZING AND BASED DRUG, OR GUIDANCE--HIGH THROUGH PUT AND ANOTHER APPROACH IS THE FRAGMENT BASED DISCOVERYY AND THIS AS PROACH HAS BEEN SUCCESSFUL IN PROPPING, ANTIPROPPINE DRUG DORPHY AND IT'S BASED ON THE NOTION YOU CAN SCREEN 11 MILLION VERY COMPLEX COMPOUNDS YOU COULD SCREEN A FEW THOUSAND OR FEW HUNDRED IN FACT, SMALLISH MOLECULES, SO LET'S SAY LESS THAN 300 WHICH ARE REASONABLE LEAK HYDROPHOBIC AND THEN FIND WEAK BINDERS OF THIS CLASS, AND YOU CAN FIND OUT BINDING STRUCTURE OF YOU TARGET MOLECULE, IT CAN MAYBE STRING THEM TOGETHER TO FORM A TITLE BINDER. SO WE HAD A COLLABORATION WITH CHRIS ABLE AND ALLISON SMITH'S GROUP AT CAME BRIDGE WHO INITIATED A PROJECT THE STUDENT IS SPEAR HEADING IN MY LAB AND SHE'S GOING BACK AND FORTH IN CAME BRIDGE TO BETHESDA, JUST LAST WEEK AND SHOWED WE CAN FIND SPECIFIC BINDING SITES AND THAT WILL BE THE SCREEN FOR COMPOUNDS WITH KBs AS WEAK, ALMOST A MILLI MOLAR. SO IT'S NOT A TIGHT BIPEDDER BUT YOU CAN IN FACT BIND SPECIFIC SITES AND I'M HIGHLY CONFIDENT THAT IN THE NEXT YEAR OR TWO THEY SHOULD BE ABLE TO STRING THESE TOGETHER AND MAYBE DEVELOP SOMETHING THAT IS AT LEAST ELITE OF A START FOR DRUG ONES AND SWITCH TO THE SECOND STORY WHICH HAS TO AFFECT THE GNP. NOW GNP IS CYCLIC MOLECULE FROM THE GNP, AND IT IS IF YOU WILL CYCLIC TUMOR RNA. EYE TINY RNA IF YOU WILL. IT'S A FACTOID, IT'S INTERESTING BECAUSE ALL BACTERIA WE KNOW EMPLOYED TO CONTROL THE LIFESTYLE. SO TO DRIVE A CHANGE IN LIFESTYLE. SO THIS MOLECULE IS LIKE A MESSENGER WHICH RESPONDS TO THE STIMULI AND DRIVES BACTERIA FOR A MOBILE PLAN TONIC LIFE FORM, TO A BIOFOAM, LIFE FORM AND LIFE FORMS I DON'T NEED TO REMIND YOU ARE VERY IMPORTANT BOTH IN THE WILD AS WELL AS IN CLINICAL SETTINGS AND IN THE CLINIC LARGE PART OF MORBIDITY A NUMBER WE CAN ESTIMATE 50% MOBILITY DUE TO THINGS LIKE CHARACTERS ARTIFICIAL VALVES AND SO ON. SO THIS SWITCH IN LIFESTYLE IS IMPORTANT BOTH PHYSIOLOGICALLY FOR BACTERIA TO SURVIVE AS WELL AS CLINICALLY AND THEREFORE THERE HAS BEEN A LOT OF INTEREST IN HOW THIS MOLECULE FUNCTIONS. AND THERE ARE MANY PROPANES WHICH INTERACT WITH THIS MOLECULE AND IN ADDITION TO MOLECULE PROPANES AND CONTROLLING THE LEVELS, BUT, WHAT CONTROLS THE DIVERSE PATHWAY SHALL REQUIRE TO TRANSFORM BACTERIA OF PLAN TONIC INNED'S HASN'T BEEN CLEAR UNTIL 206 AND RIBOSWITCH WAS FOUND. SO THIS IS THE GNP RIBOSWITCH BY THIS GROUP, REGULAR LAB AND THIS MOLECULE BINDS SPECIFICALLY TO CYCLIC GNP OF THE ORDER OF THE NANO MOLAR. AS YOU SPECT PERHAPS, THE MOLECULE WHICH CONTROL THIS IS BIG SWITCH IN LIFESTYLE, THIS RIBOSWITCH CONTROLS ALLOWS DIVERSITY OF CHANGES. SO, SAME GROUP, FOUND THAT IT IS ASSOCIATED WITH DIVERSITY OF GENES INVOLVING THINGS OF DISTIRCHGHT DIFFERENT AS [INDISCERNIBLE], SECRETION, CHEMO TAXIS, SIGNAL TRANSDUCTION, AND METABOLISM AND MEMBRANES AND SO ON. OKAY. SO DEPENDING ON THE ORGANISM AND THE BANK ACCOUNTERIAM AS MANY AS 30 COPYYS OF THIS RIBOSWITCH HAS BEEN GIVEN FOUND IN THE GENOME. SO, WE GOT INTERESTED IN THIS, FROM THE STANDPOINT AND, A FEW YEARS AGO, WE SAW THE STRUCTURE OF THE RNA IN THE LIGAND BON, AND IT'S ASSOCIATED WITH THE STEMS, IN FACT CLOSE TO EACH OTHER, CAN YOU IGNORE THIS, THIS IS JUST A CRYSTALLIZATION TRICK, WE USE THIS IN THE LAB, WE HAVE LIQUID PROPANE TO MAKE THIS CRYSTALLIZABLE. AND THIS IS FOBBED TO BE OF REDUCED [INDISCERNIBLE] OF THE CYCLIC GNP, THEY ARE NOT SINGLE STRANDED YOU CAN FORM THIS HELIX AND THE MOLECULE, THE MESSENGER IS BOUND THERE. OKAY? SO, IT IS INTERESTING PERHAPS BY PHYSICAL POINT OF VIEW IS THAT THIS IS CYCLIC LIGAND, RITE TO GNP, IDEBTICAL AND YET IT BINDS A SINGLE RNA, IN SOME WAY BREAKING SYMMETRY, BINDING ASYMMETRICAL AND THE TPT STORY I MENTIONED, THIS IS--THIS LIGAND WHICH LOOKS LIKE RNA IS BOUND LIKE RNA, MEAN TGF MAKES THIS HERE, SO THIS GENE FOR THIS CYCLIC GNP WAS NOT THERE, CONCOGNITIVICCAL AND MAKES THIS HOOK INTO WHAT'S A QUICK BASE PAIR. THAT'S ALL FINE AND WE CAN DISCUSS MORE ABOUT THE STRUCTURAL RNA, BUT THE POINT I WANT TO MAKE IS THAT IN ADDITION TO A LIGAND WHAT APPEARS TO STABILIZE THIS PACKING, AGAIN IN PRINCIPLE AND FAVORABLE PACKING OF RNA HELIXES ARE TWO LONG RANGE OR TORTEUR INTERACTIONS, THIS IS A G ONE HELIX TERM WITH A ANOTHER HE'LL 66 THERE'S INTERACTION BETWEEN WHAT IS CALLED A LOOP, A LOOP OF NUCLEOTIDES AND A RECEPTOROT OTHER SIDE. SO THIS IS THE QUESTION WHICH IS THE BALANCE STRUCTURE LIKE THE LIGAND AND IT'S CRYSTALLIZED WHAT IS THE FREE FORM OF RNA. THAT'S LESS STABLE AND HARDER TO STUDY AND WE CAN [INDISCERNIBLE] SOLUTION BY A VARIETY OF TECHNIQUES INCLUDE THANKSGIVING WHICH IS A SCATTERING AND I WON'T ENTER THE DETAIL BUS BASICALLY WHAT THIS SHOW SYSTEM THAT UPON ADDITION OF HIGH MAGNESIUM, THE PHYSIOLOGIC, HIGH MAGNESIUM, THERE ARE COMPACT A BIT BUT ADDITION OF THE LIGAND, MAKE COMPACTION OF THE RNA AND THEY CAN TAKE THE DATA AND THE [INDISCERNIBLE] AND THROUGHOUT THE PETITIONAL TRICK, DEVELOP [INDISCERNIBLE] IS OTHERS, ONE CAN REGENERATE LOWER SOLUTION ENVELOPES SHAPES AND FORMS OF THE RNA AND SOLUTION. SO THIS IS THE ENVELOPE COMPUTED FOR THE FREE FORM, UNDER THE BOUND FORM. THE BOUND FORM, THE RESOLUTION OF THESE IS [INDISCERNIBLE], FITS IN WELL WITH WHAT WE EXPECT FROM THE CRISTICAL STRUCTURE BASED FORM OF THE RNA AND IT'S OKAY. THE FREE FORM IS VERY DIFFERENT AND THE ONLY WAY WE CAN FIT THIS WITH RNA IS TO SPLIT THIS TYPE OF ARRANGEMENT AND SUCH AS THAT FACING OPPOSITE DIRECTIONS, OKAY? SO THAT'S WHAT WE CAN SAY FROM SACKS AND THIS IS HAPPENING IN THE SOLUTION IS THIS IS A STATIC THING, THIS EXISTS IN IN FORM AND LIAISON GAPPED BINE INTO THAT AND THERE'S SOMETHING ELSE MORE COMPLEX GOING ON TO LOOK AT THAT WE INITIALED COLLABBUATION WITH WAYNE'S GROUP AND HIS [INDISCERNIBLE] AND COLLABORATE WIDE US IN MEASURING THIS LARGE FORMATION USING SINGLE AND WHAT WE DO IS PUT ACCEPTORSOT TWO TIPS OF THE HELIXES AND WATCH THIS MATTER THROUGH A MICROSCOPE IN THE PRESENCE OR ABSENCE OF THE LIGAND, THAT'S WAWE CAN EXPECT FROM THE FOREGOING, THERE IS SUCH A SIGNAL AND IF YOU LOOK AT THIS CAREFULLY, CAN YOU SEE THE SIGNAL ANTICORRELATE AS YOU EXPECT FROM THE FOREGOING, BUT FOR UNEXPECTED IS THAT THE RNA IS NOT SIMPLY FLUCT WAITING BETWEEN THE OPEN AND CLOSE BUT IN FACT IT EXISTS IN FOUR DISTICKET POPULATIONS MEANING HALF AN OUR HORMONE MORE, WITH IS DURATION OF EXPERIMENT FOR BLEACH. BUT IF IT'S DOC AND DOC AND SPENDS MOST OF ITS TIME IN A DOC FORMATION IN A [INDISCERNIBLE] STATE. AND METHYLATION, DOC AND DOCS SPENDS MOST OF IT'S TIME IN DUCT FORM. THERE„i ARE TWO CONFIRMATIONS OF SUBPOPULATIONS WHICH EITHER ARE DUCT AND NOT CONDUCT IN HALF AN HOUR OR DUCT AND RARELY DUCK. SO THERE'S MORE TO„i THIS THAN SIMPLY TWO STATES. THERE'S PROBATION STRUCTURE AND WE SEE IN RESPONSE TO A LIGAND WHAT SHIFT IS THE STRUCTURAL POPULATION SO HERE IN THIS COLUMN WE HAVE THE MINUS LIGAND FROM THE RNA A LOT OF IT, AND DUCT FORM AND VERY LITTLE EXPLORES THE DUCT FORM AND THE DYNAMIC MOLECULES, ADDITION OF THE LIGAND DRIVES MOST OF THE RNA, 60% TO A STATICICAL INDUCT FORM BUT THERE'S POPULATION LEFT AND AS A CONTROL WE USE THIS MOLECULE, [INDISCERNIBLE] WHICH IS SIMILAR AND A LIGAND WHICH IS NOT BIND AND BY ASSAYS THROUGH RNA, AND YOU CAN SEE THAT COLUMN IS IDENTICAL TO THAT COLUMN AND THIS RNA SUPERVISING AND THIS CHANGE IN POPULATION IS A RESPONSE TO SPECIFIC BINDING TO TO SMALL MOLECULES. NOW WHAT DOES THIS MEAN, PHYSIOLOGICALLY, WE DON'T KNOW BUT ONE EXPERIMENT WE CAN DO IS TO INTERROGATE THAT EXPERIMENT USING WHAT THEY'VE LEARNED FROM THE KRYSTAL STRUCTURE OF THE DUCT FORM. I MENTIONED TWO INTERACTIONS, DEASHIARY INTERACTIONS THE RECEPTOR INTERACTION, THIS HELICAL BASE PAIR WHICH APPEAR TO STABILIZE THE ADULT FORM, SO WE CAN COMBINE MUTE O GENESIS WITH SINGLE MOLECULE SITE AND SEE HOW THE STRUCTURE CHANGES AS A RESULT OF THIS INTERACTION AND THAT INTERACTION. AND SO HERE IS A RESULT. SO IN THE TOP LINE HERE WE HAVE THE WILD-TYPE, OKAY, SO THIS IS MINUS LIGAND, LAST LIGAND AND THIS IS THE HIGH [INDISCERNIBLE] AND LOW [INDISCERNIBLE]. AND IN THE LIGAND, MOST OF THESE ARE IN THE LOW STATE AND SOME ARE ALREADY IN THE HISTATE AND SOME OF THE STRUCTURE SHIFTS AS I MENTIONED BEFORE. IF WE ABOLISH THIS PAIR AND MAKING MUTATION ON THIS G TO C, THIS IS NOT VISIBLE BUT THERE'S A LITTLE PICK HERE AND THE LIGAND IS A SLIGHT INCREASE OF THE POPULATION BUT IT'S MOSTLY GONE, OKAY? SO WE CONCRUITED THIS BASE PAIR IS ESSENTIAL FOR THE RNA TO EXPLORE THIS STATE, OKAY? EVEN IN THE ABSENCE OF LIGAND, THIS MUTATION,OT OTHER, THIS ELEMENT UP HERE, AND THAT SEEMS TO BE LESS DRAMATIC. YOU SEE A SMALL ONE HERE, EVEN ON THE SCALE ASK THE RNA RESPONSE IS SMALL MOLECULE MODESTLY, NOT TO THIS EXTENT BUT A LITTLE BIT, SOME BINDING AND THIS CORRELATES WITH THE SOLUTION BINDING ASSAY, WHICH I WON'T SHOW YOU TODAY. SO, WHAT THIS IS TELLING US IS THAT THE R NA INFACT IS EMPLOYING THIS TERTIARY INTERACTION WHICH IS ALOESTERRIC IN THE DISTANCE OF THE BINDING SITE TO ORGANIZE ITSELF INTO SOMETHING RESEMESTER BLELLING THE BOUND CONFIRMATION PRIOR TO LIGAND BINDING OKAY? AND WE VISUAL THE RNA EXPLORES SOMETHING SIMILAR TO THE BOUND STATE EVEN ABSENCE OF LIGAND AND WHAT WE THINKS THIS MEANS BIOLOGICALLY THEY CAN PRESENT THE ELEMENTS FOR INTERACTIONS PROPERLY AND SAYING SOME IN DIFFERENT WAY OF SAY THANKSGIVING IS THE RNA IS PREORGANIZED AND FACILITATE BINDING RAPIDLY BUT CHANGING LEVELS OF THE LIGAND BY HAVING THE BINDING SITE RECEPTED MORE PRODUCTIVELY, THAT'S HYPOTHESIS, AND WE'RE IN THE PROCESS OF MEASURING DIRECT LEAP THE LEVEL OF PREORGANIZATION OF THE BINDING SITE. BUT THE OTHER THING THIS SUGGEST SYSTEM TARGETING THE BIPEDDING SITE WITH PERHAPS DRUG LIKE COMPOUNDS THAT THEY CAN DO IS TARGET THE INTERFACE WHICH IS MUCH LARGER AND STARTED WIDE CONSERVED--SORRY CONSERVED FUNCTIONAL GROUPS AND THAT MAY PROVIDE ANOTHER AVENUE TO DISCOVERING DRUGS AND WE OF COURSE HAVE ALREADY A FRONT BASED ASSAY FOR THIS. SO WE'RE IN THE PROCESS OF THINKING ABOUT DEVELOPING THIS HAD ASSAY FURTHER TO MAKE IT A HIGH THROUGHOUT AND PUT THIS THROUGH A DRUG DISCOVERY PROGRAM. OKAY, SO THAT WAS THIS STORY AND NOW, IN THE LAST 20 OR SO MINUTES LET ME TURN TO THE ISSUE OF OKAY, WE HAVE ALL THIS RNAs IN NATURE AND I TALK ABOUT TWO OF THEM, IS THERE IN PARTICULARULARRITY TO THE WAY THEY EVOLVE AND WHAT IS THE IMPACT OF THIS ON BIOCHEMISTRY AS WELL AS OF COURSE ON ISSUES LIKE THE EVOLUTION OF DRUG RESISTANCE. SO I'LL TELL YOU ABOUT THE FITNESS LANDSCAPE OF RNA AND THAT--THAT IS ABOUT EVOLUTION, IT TURNS OUT RNA IS A PARTICULARLY NICE MOLECULE FOR STARTING EVOLUTION IN TEST TUBE, OKAY? AND THIS IS BECAUSE RNA CAN BE BOTH THE GENOTYPE AND PHENOTYPE. RNA HAS SEQUENCE AND THAT SAME MOLECULE COMES FORWARD AND THIS LINKAGE MAKE ITS POSSIBLE TO CARRY OUT THE EVOLUTION. THIS DEVELOPED IN THE 80S BYAT LEAST THREE GROUPS, JOYCE, AND GOLD. AND THE DECKNOLOGY DEVELOPED COME TO BE KNOWN AS EVOLUTION OF RNA OR SELECTS. AND THIS BASICALLY IS THE PROCESS OF SELECTS ONE MAKES A FOULING 10 TO 15 DIFFERENT SEQUENCES, DIFFERENT RNAs, MUTATION OR THROUGH CHEMICAL SYNTHESIS AND THEN THIS POOL IS PUT THROUGH SOME SELECTIVE PRESSURE. THE MOLECULES WHICH ARE ACT 95 THIS SELECTION ARE THEN AMPLIFIED THROUGH THE MAGIC OF PC R AND THIS CYCLE IS REPEATED SAY, FIVE, SIX, 10, 15 TIMES UNTIL ONE IS LEFT WITH VERY ACTIVE MOLECULES AND THERE ARE TYPICALLY VERY FEW OF THEM AND SO THAT HAS LED MANY GROUPS TO DISCOVER FOR INSTANCE, THAT IN ADDITION TO THE NATURALLY OCCURRING RIBOSIMS WHICH CATALYZE EITHER PEPTIDE FORMATION, THE RIBOSOME OR PHOSPHOR LACE TRANCE FER,--TRANSFER, THERE ARE SEQUENCES CABLE OF DOING OTHER THINGS LIKE ACTION, FORMATIONS AND MICROADDITIONS AND SO O. SO THIS HAS SHOWN THAT RNA HAS A LOT OF CHEMICAL POTENTIAL, ABILITY I SHOULD SAY. BUT THE OTHER THING THAT THIS TECHNOLOGY ALLOWS IN PRINCIPLE IS TO STUDY IN THE PROCESS OF EVOLUTION ITSELF. OKAY? SO IN THE MOST GENERAL SENSE, EVOLUTION CAN BE CONCEPTUALIZED OPTIMIZING WALK ON THIS SURFACE THAT IS CALLED A THICKNESS LANDSCAPE, OKAY? THIS IS GREWSED IN THERAPIES BY [INDISCERNIBLE] IN THIS PAPER AN ORIGINAL CONCEPTION OF EVOLUTION, WHAT HAVE YOU IN THIS IS TO HAVE DIFFERENT ALLELES FOR TWO GENES. AND IN THE THIRD DIMENSION COMING IN AND OUT OF THE SCREEN, HAVE YOU THE FITNESS, THE PHENOTYPIC FITNESS THAT CORRESPONDS TO THE PARTICULAR GENOTYPE RESULTS IN COMBINATION OF ALLELES. THIS IS WHAT THIS IS CALLED A FIELD IN THE SENSE THAT IF YOU KNOW THE SHAPE OF THIS THING, YOU CAN PREDICT EVOLUTION. BECAUSE FOR INSTANCE YOU KNOW THAT IF YOU START IN THIS VALLEY HERE YOU'LL TEND TO GO TO ONE OF THE PEAKS OR IF YOU START IN THIS PEAK, TELL BE DIFFICULT OR IMPOSSIBLE TO END THAT BECAUSE OF TRANSLATIONAL TRAVERSE VALLEY WHICH IS START NOTHING THE SUBTLE POINT, CAN YOU GO WITH THAT PEAK OR THAT PEAK. THAT'S CONCEPTUAL. SO, MINOR [INDISCERNIBLE] AND THEY HAD THIS IDEA OF EVOLUTION IN THE 70S AND THE NINOR SNPs, WHAT YOU HAVE INSTEAD OF ALLELES ARE LOCI, A AND B, IF YOU HAVE NUCLEOTIDES OR AMINO ACIDS AT POSITION ONE AND TWO AND SO ON. SO THIS IS NICE CONCEPTUALLY, BUT IN FRACTIS IT'S COMPLETELY INTRACTABLE BECAUSE HAVE YOU A TUMOR RNA, THIS IS OF COURSE YOUR SURFACE BUT IF HAVE YOU A HUNDRED MORE, HAVE YOU A HUNDRED DIMENSIONS LIKE THIS AND ONE FITNESS DIMENSION CORRESPONDS TO THAT GENOTYPE. AND THIS HAS BEEN USEFUL FOR [INDISCERNIBLE] TO TALK ABOUT IN EVOLUTION, IN PRACTICE IT HAS BEEN IMPOSSIBLE TO MAP WHAT THE SURFACE LIKE THIS LOOKS LIKE EVEN THOUGH THIS WOULD BE THE MOST POWERFUL WAY TO LOOK AT EVOLUTION. SO WHAT WE DEVISED IS AN EXPERIMENT WHICH ALLOWS US TO SAMPLE, AS YOU'LL SEE SOMEWHAT PARSLEY BY THE SAMPLE, THIS SURFACE ON RNA AND AN EXPERIMENT IS BASICALLY EELABORATION OF ONE EXPERIMENT WHICH WAS PUBLISH INDEED THIS ORIGINAL SELECT PAPER BY [INDISCERNIBLE] BACK IN 1990. THIS WAS SELECTED FOR RNA WHICH CAN BIND [INDISCERNIBLE], DOESN'T MATTER WHAT IT IS, IT'S AN [INDISCERNIBLE], IF YOU WILL, AN RNA THAT BINDS SMALL MOLECULE AND WHEN THEY DID SELECT DISPORE A DOZEN CYCLES, WHAT THEY GOT OUT OF IT, THEY GOT HAD THIS SEQUENCE, OKAY? AND TO INTERROGATE HOW THIS RNA WAS FUNCTIONING MAYBE A SIMPLE EXPERIMENT, THEY [INDISCERNIBLE], THE WINNING SEQUENCE, IF YOU WILL, TO SOME LOW LEVEL, AND DID ONE MORE CYCLE OF SELECTION, AND THEY CLONED, THE RESULTS POPULATION AND SEQUENCE 20 OF THOSE AND THEY FOUND THAT THIS POSITIONS IN BOLD HERE WERE INVARIANT AND FROM THAT THEY DID USE THAT NUCLEOTIDES ARE INVOLVING FORMING BINDING POCKET. SO THIS IS 1990 AND FAST FORWARD 20 YEARS WE CAN SEQUENCE A BIT MORE ON THIS MORE EASILY. THE EXPERIMENT WE'RE DOING IS BASICALLY TO SEQUENCE SAY A MILLION TIMES MORE USING NECKS GEN SEQUENCING AND SEE FIGURE MORE IS BETTER. I'LL SHOW YOU AN EXPERIMENT THAT WE CARRIED OUT WITH THIS MODEL SYSTEM. THE IT DOESN'T MATTER TOO MUCH WHAT IT IS, IT'S ARTIFICIAL RNA SELECTED BY THE [INDISCERNIBLE] LABS BACK IN 1995. IT'S IT IS LIGASE, RNA LIGASE WHICH TAKE THIS IS HYDROX OWN AND BIND ITS TO ITSELF FORMING A TWO PRIME, THREE PRIME [INDISCERNIBLE]. IT'S BEEN KNOWN THAT THIS BULGE HERE IN THE MIDDLE IS RESPONSIBLE FOR THE SELECTIVITY AND THE REST OF THE RNA, THIS PART, IT'S REQUIRED FOR THE RIGHT ENHANCEMENT WHICH IS 10 OF 10 OF A BACKGROUND AND WE HAVE STRUCTURAL INFORMATION ON THIS AND SO ON, BUT THIS LIGASE IN FACT IS UP WITH THE EASY SYSTEM TO PUT THROUGH OUR SELECTION EXPERIMENT BECAUSE IT LIGATES AND THEREFORE ONE CAN START WITH A SUBSTITUTE WHICH HAS A [INDISCERNIBLE] INTACT LIKE THIS AND WE CAN METRICSITAGEINIZE THE PORTION OF THE RNA AT SOME LEVEL, SAY SEVEN% FOR NUCLEOTIDES LEO TIDE FOR PERSON, IN TOTAL, AND WE CAN PUT THIS POOL OF RNAs IN THE PRESENCE OF THIS LIGAND WHICH ARE [INDISCERNIBLE], THE MONOLYTIC BEAT AND PULL OUT THE ACTIVE SPECIES, AND WE CAN TAKE THE ENTIRE THING, RT AND SEQUENCE AND SEE WHAT HAPPENS. SO THIS IS WHAT HAPPENS IN THIS CASE, I'M SHOWING YOU THE RESULT FOR 24 HOUR INCUE BATION, DEPLETION BECAUSE OF PULLING OUT THE ACTIVE SPECIES, WHAT THIS S&P IN ACCESS, IN THE ORDINATES SWREE THE NUMBER OF INDIVIDUALS OF THAT PARTICULAR SEQUENCE, ON THIS AXIS WE HAVE THE GENETIC DISTANCE FROM THE WILD-TYPE OR THE MASTER OF SEQUENCE. THE LOWEST SELECTION, POST SELECTION AND IN THE PRESELECTION POPULATION WE HAVE THREE PEAKS AND THAT'S BECAUSE THERE THERAPIST IS A COMBINATION OF THE MUTA BEGINNIZED CALCULATION HERE AND TWO POOLS WHICH WE ADD THERE AS CONTROLS WHICH SHOULD NOT BE MEANINGFULLY SELECT INDEED ONE STEP. THIS IS A RANDOMIZED POPULATION AND THIS RNA IS ABOUT [INDISCERNIBLE], SO THAT'S [INDISCERNIBLE] AND THE CALCIUM AND THIS IS A POPULATION WHICH IS RELATED TO THE MASTER SEQUENCE, A411 AND ENGINEERED IN A CERTAIN WAY, WHICH DOESN'T CONCERN US IN THIS TALK BUT THIS POPULATION IS VERY INACTIVE. SO WE EXPECT BOTH OF THESE TO BE SELECTED AGAINST IN THESE 24 INCUE BATION AND THAT'S WHAT HAPPENS, THIS IS A LOG SCALE AND CAN YOU SEE THIS IS GONE TO ZERO AND POPULATION HERE HAS MOVED TO THE RIGHT AND YOU CAN START TO SEE THE WILD-TYPE IN MASTER SEQUENCE. NOW, THIS IS IF YOU WILL LIMITATION SPECTRUM, IT'S JUST GENETICS, RIGHT? FOR THIS TO BE THIS, WE HAVE TO KNOW SOMETHING ABOUT THE PHENOTYPIC FITNESS, THE BIOCHEMICAL ACTIVITY OF EACH SPECIES, OKAY AND ONLY IF WE KNOW THAT, CAN WE TAKE THIS LEAP OF FAITH, BUT THE ABUNDANCE OF A GENO TYPE WOULD BE PROPORTIONAL TO FITNESS, IF THAT WERE TO, THEN WE CAN COUNT THE FREQUENCE OF A GENO TYPE AND GET BIOCHEMICAL INFORMATION. RIGHT? SO, JASON WAS DOING OLOGY THIS WORK IN MY LAB AND HAD TO SPEND THE NEXT FEW MONTHS OF THE PHENOTYPIC RATE CONSTANT OF ALL THE SINGLE MUTANTS NOT EVERY MUTANT BUT THE SIEGELE MUSEUMITANTS DOING HUNDREDS OF THESE THINGS AND SHOWING THEM TO SHOW YOU THAT YOU CAN RESULT CLEANLY THE INPUT FROM THE RNA AND KINETIC FOR THIS WHICH YOU DO BY RUNNING THIS OVER AND OVER AGAIN AND AT THE END OF THAT, HE HAD TO WEIGHT CONSTANT FOR ALL POSSIBLE SINGLE MUTANTS SO NOW WE CAN PLOT THE ENRICHMENT IN A PARTICULAR MUTANT, OKAY? AND MUTATIONS, MUTATION, THE MUTANT, WITH--WHICH IS HERE, WITH THE RATE MEASURED BIOCHEMICALLY AND WHAT YOU GET, ACTUALLY IS THIS LINE, THE REGRESSION IS NOT FANTASTIC, BUT IN FACT THIS, IS FAR FROM RANDOM BECAUSE WE CAN RANDOMLY REASSORT THESE TWO VARIABLES, YOU GET DISTRIBUTION OF OUR VALUES AND OUR MEASURED ARM IS WAY OUT HERE AT 7.4, SEVERAL DEVIATIONS AND THIS IS HIGHLY NORMAL. NOT VERY POWERFUL, BUT WE CAN SAY, WITH CERTAINTY THAT IN FACT, THE FREQUENCY OF A MUTANT IN THE SELECTIVE FORM, IN FACT IS A QUANTITATIVE MEASURE OF IT'S BIOCHEMICAL ACTIVITY. SO, THEN WE HAVE IN FACT, SAMPLED SPARSELY, AN RNA LANDSCAPE, SO THIS PLOT, CAN YOU IGNORE THESE WHICH ARE VERY FAR FROM THE WILD-TYPE WHICH IS HERE UNDERSTANDING DISTANCE, THIS IS JUST ARTIFACTS BUT THIS PART HERE IS A LANDSCAPE AND THAT'S COLORED MOLECULES AND GENOTYPE WHICH IS ARE SLOWER, IN PURPLE AND FASTER OR FAST I SHOULD SAY IN GREEN. SO THIS IS--THIS PLOT RESULTS IN SUBTRACTING THE FREQUENCY OF WITH FAST SHORT SELECTION OR THEY'RE ALLOWED TO LIGATE FOR ONE MINUTE, FAST MOLECULES VERSES THOSE WHICH TAKE 24 HOURS TO LIGATE. AND WHAT IS INTERESTING IS THAT THIS IS EXTREMELY STEEP OR RUGGED, TO USE THE TERMINOLOGY FOR EVOLUTION AND WHAT IT MEAN SYSTEM THAT WE CAN MAKE SMALL WALKS IN GENETIC SPACE AND GO FROM REALLY FAST TO REALLY SLOW AND REALLY FAST AND THAT IS INTERESTING FROM EVOLUTIONARY POINT OF VIEW BECAUSE YOU MIGHT ARGUE IN FACT IT IS DIFFICULT FOR RNA TO MUTATE WITHOUT LOSING FUNCTION AND WE'LL COME BACK IN A MINUTE, AGAIN, BUT NOTHING YOU CAN DO WITH THIS KIND OF THING IS TO OF COURSE ANALYZE THE LANDSCAPE IN DIFFERENT WAYS. ONE THING WE'VE BEEN DOING IS TO USE THIS KIND OF ANALYSIS WHICH IN PRINCIPLE TAKES ONE AFTERNOON TO ASK IN ONE EXPERIMENT THE FUNCTIONAL IMPORTANCE OF ALL NUCLEOTIDES OF RNA AND WHAT WE'RE APPROACHING HERE IS THE INFORMATION CONTENT WHICH IS A MEASURE OF SEQUENCE CONSERVATION OF OUR ABILITY AFTER ANY GIVEN POSITION OF RNA FOR FAST LIAISON GATING MOLECULES THIS, IS ACTIVE MOLECULES VERSES SLOW ONES, CAN YOU SEE THERE'S QUITE A BIT OF DIFFERENCE FOR INSTANCE THIS A'S IMPORTANT FOR BEING FAST BUT NOT SO IMPORTANT FOR BEING SLOW AND SO ON. IF YOU SUBTRACT THIS FROM THIS, YOU GET THIS, WHICH IS A CHANGE IN INFORMATION CONTENT FROM SLOW TO FAST RNAs, IN OTHER WORDS THIS IS THE SLOPE OF THE FITNESS LANDSCAPE WHICH R NA HAS TO TRAVERSE TO BECOME MORE ACTIVE AND SO AT LEAST FOR THIS, IF YOU'LL TOY WITH THE EXAMPLE, WE HAVE TECHNOLOGY THAT ALLOWS US IN ONE EXPERIMENT TO COUNT FOR THE GENOTYPES WE HAVE SHOWN THAT THE ABUNDANCE OF THE SEQUENCE THAT COMES THROUGH THE SELECTION IS IN FACT A GOOD SURROGATE FOR THICKNESS AND WE CAN INFACT DO THAT CHEMISTRY. QUANTITATIVELY SEQUENCING. THE LIMITATIONS AS USUAL, WITH THE CURRENT LYM NA--LYM NA, SEQUENCING SECNOLOGY WE ARE REALLY LIMITED TO 200 NUCLEOTIDES, A HUNDRED NUCLEOTIDES WE CAN DO A SINGLE READ AND THAT'S USING EACH INDIVIDUAL, SO IT REALLY CANNOT TOLERATE MUCH IN THE WAY OF SEQUENCING ERROR AND EVEN WITH THIS TECHNOLOGY WE CAN ONLY SEQUENCE ABOUT 10 TO EIGHT SEQUENCES AND AS I MENTIONED, THE EXPERIMENT STARTS WITH MAYBE 10 TO 15 SEQUENCES SO WE'RE STILL UNDERSAMPLING BY MANY, MANY, LOGS. NONETHELESS, THE SEQUENCE SUGGEST GETTING CHEAPER EVERY DAY. RNA CSR DNA, PEPTIDES THAT ARE SHORT ENOUGH, AND I WOULD PAUSE FOR ANY SYSTEM THAT IS GENERATED AND THEN CALLED, THE IMMUNE SYSTEM COMES TO MIND VUBSLY BUT THINGS LIKE [INDISCERNIBLE], VIRUS EVOLUTION AND IN GENERAL ANY GENETIC CONFLICT AS LONG AS IT CAN BE DEFINE WIDE A SHORTAGE SEGMENT YOU CAN ANALYZE. AND, I MOVE TO MAKE A PLUG HERE, IT TURNS OUT THAT MOST OF THE ALUMINA SOFTWARE IS GEARED TOWARDS DOING SNIPS OR SHOTGUN SEQUENCING SO WE HAVE TO WRITEOT SOFTWARE TO ALIGN EACH SEQUENCE BUT [INDISCERNIBLE]. IN FACT, THOSE ARE FROM THE BELIEVER IN OPEN SOURCE THIS, IS AVAILABLE IN SOURCE [INDISCERNIBLE] AND I HOPE THEY CAN USE, THIS IS ENTIRE DATA SET AND PRETTY EFFICIENT. SO LET ME FINISH BY TAKING THIS NOTION OF THE CONCEPT OF EVOLUTION OF DRUG RESISTANCE AND HOW WHAT WE HAVE LEARNED AT LEAST FOR THIS RTHIS,A IMPACTS THAT. THIS IS THE LANDSCAPE AS I MENTIONED, MUTATION, SMALL CHAIRVELG CHANGES IN--CHANGES IN SEQUENCE, RESULTS IN DRASTIC MOTILITY AND THAT IS WITH STRUCTURE WHICH IS STABILIZED IN MANY LOCATIONS BY SINGLE BASE PAIRS. AND SO AS AN EXAMPLE I TALKED ABOUT CYCLIC GNP, AND ONE HELIX BASE WILL BE LOST AND RESULT IN LOSS OF ACTIVITY. SO ONE THING IS THAT YOU MAY WONDER HOW RNA EVOLVED AND WE HAVE AN ANSWER TO THAT WHICH I WON'T DELVE INTO TODAY BUT ALTHOUGH FOR A GIVEN FUNCTION FOR IT TO BE STEEP, IT TURNS OUT THAT SEQUENCE CLOSE IN GENETIC SPACE CAN HAVE DIFFERENT FUNCTIONS AND WE THINK RNA ACQUIRING NEW FUNCTION WHICH IS IS COMPATIBLE IN BIOLOGY BUT IN A DRUG KIND OF CONTEXT IS ALSO IN FACT GOOD NEWS BECAUSE I WOULD POSE IT THAT YOU CAN MAKE RNA DRUGS THAT WILL BE NOT SO TRIVIAL FOR RNA FOR A SINGLE AND DOUBLE MUTATION TO ESCAPE THE EFFECTS OF THAT DRUG. SO I THINK THE PROSPECTS ARE BRIGHT BUT I WILL WILL HAVE TO SHOW THAT EXPERIMENT. SO, LET ME THANK SOME PEOPLE. TOM EDWARDS, [INDISCERNIBLE], AND COLLABORATORS IN CAMBRIDGE, ALLISON SMITH, AND OTHERS INVOLVE WIDE THE RIBOSWITCH PROJECT. NATHAN [INDISCERNIBLE] COLLABORATE WIDE ME ON THE CYCLIC GNP PROG EXPECT WE HAVE AN ONGOING COLLABORATION WITH DEBSCHECHARLOTTE, AND [INDISCERNIBLE] WITH THE SAME RNA. THIS ANALYSIS WAS REALLY DRIVEN ENTIRELY BY JASON PIT WHO WAS A FORMER VERY TALENT TED POST DOC IN THE LAB AND [INDISCERNIBLE] HELPED US WITH THE MATHEMATICAL ANALYSIS OF THE MOUNTAIN OF DATA THAT KIND OF THING GENERATES. THESE PEOPLE FUNDED US, I'LL BE HAPPY TO TAKE QUESTIONS AND I THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ] >> QUESTIONS? >> SO YOU LOOK AT THE SINGLE MUTATION LANDSCAPE AND IT SEEMS TO EVEN THE [INDISCERNIBLE] AND PERHAPS LOCATING SOME OF THE BASIS BASE PAIRS ARE MORE IMPORTANT THAN THE OTHERS DID YOU LOOK AT THIS. >> YES, ABSOLUTELY, SO NOT ALL BASE PAIRS ARE EQUAL, SOME MORE EQUAL THAN OTHERS, RIGHT? SO YEAH, CAN YOU SEE THIS EXAMPLE, WHERE FOR INSTANCE, THESE NUCLEOTIDES HERE WHICH ARE IN PURPLE AREENTIOUS SENTIALLY NO IMPACT ON THE FUNCTION WHERE SOME OTHERS WHICH ARE OTHER COLORS LIKE THIS ONE OR MAYBE THIS I SUSPECT THERE'S A GAPPAR HERE, THESE TWO ARE VERY IMPORTANT AND SO YES, IT'S EQUAL AND IN FACT, YES, RNA COULD EVADE PERHAPS DRUGS BY EVOLVE THANKSGIVING DISTANT REGIONS BUT ALSO THE FUNCTION IS LESS IMPORTANT, SO THE SETTLIZATION OF THE SECONDARY STRUCTURE MAY LEAD TO SOME ESCAPEES BUT I DON'T THINK IT'S A HUGE PROBLEM IN THE WAY IN SENSE THAT MANY OF THESE SINGLE NUCLEOTIDES ARE IN THAT SIDE AND THE IDENTITY ASK, EXACT IDENTITY MATTERS A LOT. WE CAN ONLY HAVE THREE POSSIBLE ALFERNATIVES NOT 19 OR 20 OR 21. SO I STILL WOULD ARGUE ON THESE GROUNDS, THAT IN PRACTICE IT WILL NOT BE SO EASY FOR RNA„i TO ESCAPE NEFARIOUS CHEMICAL. [INDISCERNIBLE]. >> SO THE QUESTION I BELIEVE IS WHAT ARE THE PROSPECTS OF COMPUTATIONALLY AND I THEN IS GOING TOWARDS THINGS FOR MIKE O DYNAMICS PERHAPS AND TO PREDICT PERHAPS THE STRUCTURES RNA IN BOUND FREE CATALYTIC AND FORMS AND SO OWELL THE PROSPECTS I REALLY CAN'T COMMENT ON BUT IN PRACTICE IT HAS BEEN VERY DIFFICULT. OKAY? SO, THERE IS OF COWER A PARALLEL [INDISCERNIBLE] IN THE PROTEIN PREDICTION FIELDS, RIGHT, AND THERE IT HAS BEEN THIS COMPETITION CALLED CAST WHERE PROTEIN FOR PREDICTER GROUPS WILL SUBMIT PREDICTIONS OF A GIVEN PROTEIN STRUCTURES ARE KNOWN YET TO THIS COMPETITION AND ONCE THE STRUCTURE BECOMES KNOWN, THEY SCORE THE RESULTS AGAINST THE PREDICTION, THE REAL STRUCTURE AND YOU CAN ARGUE WHETHER IF IT'S GOOD OR BAD, THEY BOTH SAY THERE IS NOW AN EQUIVALENT EFFORT IN RNA FIELD AND I CAN TELL YOU THAT FROM THE FIRST ITERATIONS WHICH JUST HAPPEN THIS YEAR, AND IF IT'S A DUPLEX, YOU ARE GOOD WITH THE STRUCTURE. AND ANOTHER ONE. AND THIS I THINK IS IN FACT PERHAPS A MORE DIFFICULT PROBLEM THAN PROTEIN STRUCTURE PREDICTION, SIMPLY BECAUSE YOU HAVE MUCH FEWER BASTERS WHO ARE MUCH FEWER INTERACTIONS THAN HAVE YOU BEASTERS YOU HAVE EVEN IN NONCLINICAL BASTERS, YOU ARE LOOKING AT BASE INTERACTION WHERE YOU SEE THE INTERACTIONS THAT ARE MUCH MORE RICH, SO PERHAPS IT IS EASIER TO FIND UNIQUE FOLDING SOLUTIONS FOR PROPANES COMPUTATIONALLY, WITH THINGS LIKE THESE ARE RNAs, IT'S A PROBLEM, IT HAS NOT BEEN EASY, AND WELL, YES. >> SO TALKING ABOUT NEEDING A PREFOLDED STATE, SO I WAS WONDERING IF IT'S POSSIBLE TO IDEBT DENTIFY RESIDUES IN THAT STRUCTURE THAT AFFECT THE KINETICS BUT NOT THE STABILITY OF THE FINAL STATE AND MIGHTAST THE RATE AT WHICH YOU ACHIEVE THIS PREFOLDED STATE. AND IF SO, WHETHER THOSE MUTATIONS HAVE PHENOTYPE IN THE ORGANISM? >> VERY GOOD QUESTION. WE DON'T HAVE CLEAN SEPARATION OF FUNCTION MUTANTS. I SHOWED YOU ONE MUTATION WITH THE RECEPTOR WHICH AFFECTS BINDING SOMEHOW, BUT DOESN'T AFFECT KINETIC SO MUCH, THOSE ARE POOR EXAMPLES, SO I AGREE IT'S POOR FUNCTION AND MIGHT HAVE AND IT WOULD BE A GREAT TOOL, SO WE HAVEN'T FOUND IT TO EXECUTE IT. >> [INAUDIBLE QUESTION FROM AUDIENCE ] >> RIGHT. AND SO, I THINK THE HIGHWAY POT SIS STILL REMAINS TRUE IN THE MOST STABLE FORM, OKAY, IF YOU WILL, THE BOUND FORM OF THE RNA WHICH BY [INDISCERNIBLE], BUT ALSO BY [INDISCERNIBLE] SEEMS TO EXIST IN A LIMITED MONEY AND FULL STRUCTURE WHICH IS ARE CLOSE TO EACH OTHER. THE ISSUE ABOUT THE PARSENESS OF THE CONTEXT IS DEFINITELY TRUE AND THAT IS IN RELATION TO PROTEINS BECAUSE IN PRACTICES AND REALITY, RNA IS--HAS NOT THE SIMPLE SECONDARY TERTIARY, QUADRANTARY BREAK DOWN WHERE YOU HAVE HELIXAL STRANDS WHERE YOU HAVE SECOND STRUCTURE AND THEN HAVE YOU A TERTIARY STRUCTURE AND HELIXES ARE STABLE [INDISCERNIBLE] SO THOSE THINGS ARE IF YOU WILL HAVE A CORE, WHICH IS THE BASE STACK AND THOSE FORM FAST AND THEY'RE PRETTY STABLE IN ISOLATION AND THE INTERACTION OF THESE HELICAL DOMAINS AND WEEK AND SPARSE. BUT NONETHELESS, THERE ARE AT LEAST IN THE MOST STABLE FORM UNIQUELY DETERMINED BY THE SEQUENCE SO IT'S STILL USING BIOPOLYMER BUT THE STABILITY ORDER SECONDARY AND TERTIARY WAS UPSIDE DOWN FROM PROTEINS WHERE IN PROTEIN FIST YOU TAKE A HELIX OUT OF CONTEXT, IT WILL MELT. AND TERTIARY STRUCTURES ARE WEAKER. SO-- >> [INAUDIBLE RESPONSE FROM AUDIENCE ] >> SELECT THE SUBPOPULATION FOR THE FAVORABLE DISTRACTION? >> RIGHT, SO THERE ARE EXAMPLES FOR EVERY CASE, BUT ON AVERAGE I WOULD SAY THAT FOR RNAs WHICH HAVE REASONABLY UNIQUE FOLDED STATE BY NMR, THE KRYSTAL STRUCTURES ARE MORE OR LESS THE SAME AND OCCASIONALLY IT'S POSSIBLE TO TRAP SOME RNA WHICH EXISTS IN MULTIPLE STATES AND SOLUTION IN ONE PARTICULAR CONFIRMATION, BUT ON AVERAGE I WOULD SAY THAT IF RNA EXIST INDEED SOLUTION IN MULTIPLE STATES IT WILL NOT CRYSTALLIZE. SO I WOULD USE THE CRYSTALLIZATION IN REVERSE, THIS ARGUMENT FOR EXISTENCE OF SOME STABLE FORMS OF SOME RNAs. OKAY. >> SO LET'S THANK ADRIAN FOR A SPECTACULAR TALK. [ APPLAUSE ] AND I'M SURE IF YOU HAVE QUESTIONS THAT YOU'RE TOO SHY TO ASK AT THE MICRO PHONE, HE WILL STAY FOR A WHILE TO ANSWER THEM.