<p>Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.</p><p> A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.</p><p> The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.</p><p> To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.</p> / Dissertation

Prostate cancer represents a serious health concern as the most diagnosed form of cancer in men and the second leading cause of cancer death in the Western world. Current treatments for prostate cancer are non-targeted and result in a number of undesirable, non-specific effects, highlighting the need for novel, targeted therapeutics in the treatment of prostate cancer. Prostate Specific Membrane Antigen (PSMA) offers great promise in the targeting of prostate cancer for imaging and therapy. PSMA is a transmembrane carboxypeptidase with cell surface expression several orders of magnitude higher in cancerous prostatic epithelia than found in other tissue and PSMA is constitutively internalized into cells. The unique expression profile of PSMA and its constitutive internalization offer great value in the targeted delivery of therapeutics to prostate cancer cell. In 2002, two synthetic RNA ligands, aptamers, were selected for their ability to inhibit the enzymatic activity of PSMA. In 2006, the utility of these aptamers in the delivery of cytotoxic siRNA across the cell membrane was demonstrated in vivo using aptamer-siRNA chimeras. However, those experiments were performed by intratumoral injection, and systemic administration will be necessary for use in the clinic. In this thesis, we improve PSMA targeted chimeras to serve as more powerful therapeutics in the treatment of prostate cancer. We optimize existing aptamer-siRNA chimeras for increased potency and stability and improved pharmacokinetics to enable systemic administration. We truncate the PSMA binding aptamers for amenability to large-scale chemical synthesis. With emerging roles for PSMA enzymatic activity in the prostate cancer disease we identify aptamers that are suitable for chemical synthesis and retain inhibitory properties against PSMA. Finally, we assess the use of aptamers as synthetic ligands in the functional inhibition of PSMA mediated motility in prostate cancer. Our results demonstrate the ability of aptamer-siRNA chimeras to specifically kill PSMA-expressing cells with cytotoxic siRNA upon systemic injection. We confirm a newly reported role for PSMA in the promotion of cell motility and demonstrate the ability of aptamers to effectively neutralize PSMA-mediated motility. The results presented within argue strongly for the functional utility of aptamers in the treatment of prostate cancer.

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed. The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve ngstrm-level resolution through careful design to reduce noise.

<p>Thrombosis, or pathological blood clot formation, is intimately associated with cardiovascular disease and is the leading cause of morbidity and mortality in the western world. Antithrombotics are commonly prescribed as prophylactic medications or as rapid onset anticoagulants in acute care clinical settings. Although a number of antithrombotics are clinically available, their use is limited by immunogenicity, toxicity, and inability to be controlled with an antidote in the event of hemorrhage. Therefore, new antithrombotics that are effective, yet can be rapidly controlled are urgently needed. </p><p>Aptamers are oligonucleotides that form complex secondary and tertiary structures based on intramolecular base pairing and nucleic acid folding that allows them to bind to molecular targets with high affinity and specificity. Aptamers can be isolated that bind to proteins, such as clotting proteins, and modulate protein function. However, unlike most currently used antithrombotics, aptamers can be directly controlled with an antidote and therefore represent a safer class of therapeutic agents. </p><p>To generate a novel anticoagulant, we developed an aptamer-antidote pair against prothrombin. Prothrombin is a blood protein that plays an essential role in clot formation. I truncated, optimized, and studied the mechanism of an aptamer that can bind to prothrombin and inhibit prothrombin function, thereby severely impeding clot formation. Moreover, to increase the safety profile of this anticoagulant aptamer, I developed an antidote that can quickly reverse aptamer function and restore normal clotting. This aptamer and antidote pair is the first antidote reversible anticoagulant that targets prothrombin and may prove to be a valuable clinical anticoagulant.</p><p>A number of anticoagulants are in development, and a wide debate regarding the optimal protein target for anticoagulation is underway. We have previously generated anticoagulant aptamers to human coagulation factor VII, factor IX, factor X, and prothrombin. I compared the effects of these four anticoagulant aptamers to determine their impact on thrombin generation and clot formation. Each aptamer exerts its own unique effect on thrombin generation/clot formation, depending on the role that its protein target plays in coagulation. These studies provide valuable data regarding target validation and the anticoagulant effects of different therapeutic aptamers.</p><p>Robust anticoagulation is required during acute clinical surgical procedures to treat thrombosis. Currently used anticoagulants have several untoward side effects and most are not antidote controllable. I tested the effects of combining two anticoagulant aptamers to assess potential drug synergy. Several combinations of two anticoagulant aptamers were synergistic and severely impaired blood clot formation. One specific pair of aptamers that targeted factor X (FX) and prothrombin in combination was extremely potent and could keep blood fluid in an ex vivo model of extracorporeal circulation. Additionally, this pair of aptamers could be functionally modulated with two different types of antidotes. In conjunction with antidote reversal, this strategy of combining aptamer anticoagulants may prove useful in a variety of highly prothrombotic acute clinical settings. </p><p>Finally, to explore the potential of aptamers to regulate platelet function, I isolated and characterized an aptamer toward platelet glycoprotein VI. Glycoprotein VI is a platelet surface receptor that plays a key role in platelet activation and platelet plug formation. I isolated several aptamers that bind to glycoprotein VI, and show that the lead aptamer binds to platelets with high affinity and causes platelet activation and aggregation. This aptamer could potentially be further developed for topical administration to manage bleeding, or for biomarker detection of soluble glycoprotein VI in patient plasma.</p> / Dissertation

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

In the last years, DNA arrays have attracted increasing attention among medical diagnosis and analytical chemists. The broad range of application that has been found for DNA arrays makes them an important analytical tool. DNA arrays are relevant for the diagnosis of genetic diseases, detection of infectious agents, study of genetic predisposition, development of a personalised medicine, detection of differential genetic expression, forensic science, drug screening, food safety and environmental monitoring.Despite the great promise of DNA arrays in health care and their success in medical and biological research, the technology is still far away from the daily use in the clinic and even more far away from their implementation in home-diagnosis such as glucose biosensors. Their principal problems are the high cost and difficulty of use, because it is required costly laboratory instruments and biology knowledge for the labelling of the DNA prior to the sample injection into the array.On the other hand, the requirements that a biosensor should include are to be easy-to use so that it do not need the previous label of the sample and the addition of reagents. It should give a sensitive response in short time, and it should also include cheap generic multi-analyte detection.The work carried out in this thesis describes new concepts of electrochemical biosensoric platforms based on oligonucleotides for detection of label-free DNA and protein, which include these requirements.Preliminary experiments of direct DNA electrochemical detection of labelled ssDNA were performed to establish a protocol of DNA immobilisation, hybridisation and detection colourimetrically and electrochemically. DNA real samples and multi-analite detection on an array developed by biocopatible photolithography were used.To avoid the analyte labelling to develop an easy to use and low cost device, a label-free electrochemical displacement of DNA sensor was described. The method of detection by displacement requires the pre-hybridisation of the capture probe immobilised on the electrode surface with a sub-optimum mutated oligonucleotide labelled with a redox molecule. Due to the higher affinity of the target that is fully complementary to the capture probe, the sub-optimum label can be displaced when the complementary target is introduced in the system. The decrease of the signal would verify the presence of the target and should be proportional to its concentration. Sub-optimum hybridisation displacement detection was demonstrated colourimetrically and electrochemically with a sub-optimum mutated oligonucleotide labelled with horseradish peroxidase (HRP), and a ferrocene sub-optimum mutated oligonucleotide was also detected electrochemically, which do not required the addition of reagents for its detection.Furthermore different strategies to develop an electrochemical oligonucleotide (aptamer) based sensor for reagentless and label-free protein detection was carried out. The most sensitive aptasensor achieved 30 fM of detection limit in just 5 minutes. / En els últims anys, els xips d'ADN han atret una atenció creixen en els camps de la diagnosis mèdica i la química analítica, degut a la seva portabilitat, sensibilitat, especificitat, ràpida resposta i l'ampli ventall d'aplicacions. Els xips d'ADN són rellevants per la diagnosis de malalties genètiques, detecció d'agents infecciosos, estudis de predisposició genètica, desenvolupament de medicina personalitzada, detecció d'expressió genètica diferencial, medicina forense, exploració de medicaments, seguretat alimentaria, defensa militar i monitorització mediambiental. Encara que els xips basats en oligonucleòtids per la detecció d'ADN i proteïnes siguin una gran promesa en medicina i recerca biològica, aquesta tecnologia es encara molt lluny del seu ús diari en el camp clínic i encara més lluny de poder ser comercialitzada per ús domèstic com ho han estat el biosensors de glucosa. Els seus principals problemes són el seu alt cost i la seva dificultat d'ús. Ja que per la seva utilització és necessari, previ a la injecció de l'analit en el biosensor, costosos instruments de laboratori i tècnics especialitzats en bioquímica pel marcatge i amplificació de les mostres d'ADN. En canvi els requeriments que un biosensor ha d'incloure són, ser fàcil d'utilitzar, per tant que l'analit no necessiti un marcatge previ i l'addició de reactius per la seva detecció. Aquest ha de donar una resposta ràpida i sensible a baix cost i ha de permetre la detecció en el mateix equip de diferent tipus d'analits.El treball fet en aquesta tesis descriu el desenvolupament de nous concepte de plataformes biosensòriques electroquímiques basades en oligonucleòtids per la detecció d'ADN i proteïnes no marcades prèviament, els quals inclouen aquest requeriments. Experiments preliminars per la detecció de l'hibridació d'ADN marcat es van portar a fi per tal d'establir un protocol per la immobilització, hibridació i detecció d'ADN colorimètricament i electroquímicament. És van utilitzar mostres reals d'ADN i sistemes de detecció de multi-analits en un xip desenvolupat per fotolitografia biocompatible.Per tal de no necessitar un marcatge previ de la mostres d'ADN i així simplificar i reduir el cost del futur biosensor es va desenvolupar un sistema electroquímic de desplaçament. El mètode lliure de marcatge es basa en el desplaçament de molècules d'oligonucleòtid mutat i marcat, els quals encara que continguin certes mutacions són capaços d'hibridar amb la sonda d'oligonucleòtid immobilitzat, però quan aquestes es troben en presència de l'analit desplaça la molècula mutada i marcada, disminuint així la senyal de manera proporcional en la concentració del analit. El sistema de desplaçament ha estat demostrat colorimètricament i electroquímicament utilitzant un marcatge d'HRP sobre el mutat i utilitzant un marcatge de ferrocè en l'oligonucleòtid mutat per tal de no necessitar afegir cap reactiu per la detecció de l'analit, També es van portar a fi diferents estratègies per desenvolupar un biosensor electroquímic basat en oligonucleòtids (aptamers) per la detecció de la proteïna trombina sense el previ marcatge d'aquest analit i sense necessitat d'afegir reactius per la detecció del analit. En el sistema mes sensible es va obtenir un límit de detecció de 30 fM en un temps de resposta de sols 5 minuts. / En los últimos años, los chips de ADN han atraído una atención creciente diferentes campos, debido a su portabilidad, sensibilidad, especificidad y rápida respuesta. Los chips de ADN son aplicados en diagnosis de enfermedades genéticas, detección de agentes infecciosos, estudios de predisposición genética, desarrollo de medicina personalizada, detección de expresión genética diferencial, medicina forense, exploración de medicamentos, columnas de separación, seguridad alimentaría, defensa militar y monitorización medioambiental. Aunque los chips basados en oligonucleótidos para la detección de ADN y proteínas tienen un gran futuro en diagnosis e investigación biológica, esta tecnología está aun muy lejos de su uso diario en el campo clínico y aun mas lejos de poder ser comercializado para uso doméstico como lo han sido los biosensores de glucosa. Sus principales problemas son su alto coste y su dificultad de uso. Para su utilización es necesario, previo a la inyección del analito en el biosensor, costosos instrumentos de laboratorio y técnicos especializados en bioquímica para el marcaje y amplificación de las muestras de ADN. En cambio los requerimientos que un biosensor ha de incluir son, ser fácil de utilizar, por tanto el analito no ha de necesitar un marcaje previo ni la adición de reactivos para su detección. Este ha de dar una respuesta rápida y sensible a bajo coste y ha de permitir la detección en el mismo equipo de diferentes analitos.El trabajo hecho en esta tesis describe el desarrollo de nuevos conceptos de plataformas biosensóricas electroquímicas basadas en oligonucleótidos para la detección de ADN y proteínas no marcadas previamente, los cuales incluyen estos requerimientos.Experimentos preliminares para la detección directa de la hibridación de ADN marcado se llevó a cabo para establecer protocolos para la inmovilización, hibridación y detección de ADN colorimétricamente y electroquímicamente. Se utilizaron muestras reales y sistemas de detección de multi-analitos en un chip desarrollado por fotolitografía biocompatible.Para no necesitar un marcaje previo de la muestra de ADN y así simplificar y reducir el coste del futuro biosensor se desarrolló un sistema electroquímico de desplazamiento. El método libre de marcaje se basa en el desplazamiento de moléculas de oligonucleótido mutado y marcado, el cual aunque contenga ciertas mutaciones es capaz de hibridar con la sonda de oligonucleótido inmovilizado, pero cuando estas se encuentran en presencia del analito desplaza la molécula mutada, disminuyendo así la señal de manera proporcional a la concentración del analito. El sistema de desplazamiento ha sido demostrado colorimétricamente y electroquímicamente utilizando marcaje de HRP sobre el mutado, así como un marcaje de ferroceno que no requiere la adición de reactivos para su detección. También se llevaron a cabo diferentes estrategias para desarrollar un biosensor electroquímico basado en oligonucleótidos (aptámeros) para la detección de trombina sin el previo marcaje de este analito, ni la adición de reactivos para la detección del analito. En el sistema más sensible se obtuvo un límite de detección de 30 fM en un tiempo de respuesta de solo 5 minutos

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

Although nucleic acids primarily function as carriers of the genetic information in biology, their chemical versatility, replicability and programmability render them much more functions inside and outside of cells. Numerous nucleic acid catalysts (known as ribozymes and deoxyribozyme) and binding agents (known as aptamers) have been engineered through the combination of directed evolution and rational design. However, new technologies and theoretical frameworks are still in need to better engineer and utilize these functional nucleic acids in diagnostics and therapeutics. Aiming at engineering more powerful aptazyme-based genetic regulators, we first devised a scheme for direct selection of physiologically active ribozymes in mammalian cells. Model-driven analysis of the selection process showed that the stringency of the selection was strongly influenced by system variables such as degradation rate of un-reacted ribozymes. This analysis led to models that can be exploited to understand and predict the performance of aptazyme-based biosensors and genetic regulators. Several fundamental limitations of aptazymes-based systems were identified from the analyses of these models. As it became apparent that the signals generated by aptazymes need to be processed and amplified at molecular level to have satisfactory effects on the final readouts, we turned our focus to engineering nucleic acid-based signal processors using several newly invented schemes such as ‘entropy-driven DNA amplifier’ and ‘catalyzed DNA self-assembly.’ We first demonstrated a method to couple entropy-driven DNA amplifiers to allosteric deoxyribozymes, and then proved that the concept of catalyzed DNA self-assembly can be used to design efficient and versatile signal amplifiers for analytical applications on various platforms. These developments may potentially lead to sensitive, low-cost, and point-of-care diagnostic devices. Taken together, these works not only addressed several important issues regarding the engineering and application of nucleic acid catalysts, but also revealed a new theme in molecular engineering: In order to better engineer and utilize a part, one needs to characterize, model, and modify the system surrounding the part so that the potential of the part can be maximized. / text