Optimization of Protein-Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy

Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio (SNR) for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases (FAK) with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput

Synthetic biology devices for in vitro and in vivo diagnostics

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.United States. Defense Threat Reduction Agency (Grant HDTRA1-14-1- 0006)United States. Office of Naval Research. Multidisciplinary University Research InitiativeUnited States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060)Wyss Institute for Biologically Inspired EngineeringHoward Hughes Medical Institut

DNA nanostructures: A versatile lab-bench for interrogating biological reactions

At its inception DNA nanotechnology was conceived as a tool for spatially arranging biological molecules in a pro- grammable and deterministic way to improve their interrogation. To date, DNA nanotechnology has provided a versatile toolset of nanostructures and functional devices to augment traditional single molecule investigation approaches – including atomic force microscopy – by isolating, arranging and contextualising biological systems at the single molecule level. This review explores the state-of-the-art of DNA-based nanoscale tools employed to enhance and tune the interrogation of biological reactions, the study of spatially distributed pathways, the visu- alisation of enzyme interactions, the application and detection of forces to biological systems, and biosensing platforms

Nanotechnology-based strategies in diagnostic and therapeutic nanodentistry

Περίληψη Η νανοτεχνολογία έχει εξαπλωθεί γρήγορα σε όλους τους τομείς της επιστήμης, παρέχοντας σημαντικές εναλλακτικές προσεγγίσεις σε επιστημονικά και ιατρικά ερωτήματα και προβλήματα. Η νανοτεχνολογία έχει επίσης τεράστιο αντίκτυπο στη βιομηχανία της υγείας και η χρήση της έχει ωφελήσει όχι μόνο τη σύγχρονη ιατρική αλλά και την οδοντιατρική. Προβλέπεται να εμπλουτίσει και επαναπροσδιορίσει περαιτέρω την οδοντιατρική, με πιθανές εφαρμογές σε όλες τις πτυχές των στοματικών παθήσεων, στην οδοντιατρική διάγνωση, πρόληψη και θεραπεία. Η επιστήμη των υλικών στην οδοντιατρική έχει ενσωματώσει τεχνολογίες για τη δημιουργία νανοϋλικών, που χρησιμοποιούνται σε ποικίλες οδοντιατρικές εφαρμογές. Αυτές οι αναδυόμενες τεχνολογίες έχουν τη δυνατότητα να προσφέρουν σημαντικά οφέλη με τη μορφή καινοτομιών στην οδοντιατρική επιστήμη και κοινωνία [Sharan et al., 2017]. Οι κατασκευαστές έχουν συμπεριλάβει μια ποικιλία νανοσωματιδίων σε οδοντιατρικά υλικά προκειμένου να βελτιώσουν τις χημικές και φυσικές ιδιότητες των υλικών αυτών. Αυτό το πεδίο έρευνας έχει τον τίτλο «νανο-οδοντιατρική» [Padovani et al., 2015]. Η νανοτεχνολογία έχει χρησιμοποιηθεί για την ανάπτυξη οδοντιατρικών υλικών αποκατάστασης με σημαντική επιτυχία. Οι πρόσφατες εξελίξεις στη νανο-οδοντιατρική και καινοτομίες στις διαγνωστικές, προληπτικές και θεραπευτικές μεθόδους που απαιτούνται για τη διατήρηση και απόκτηση της τέλειας στοματικής υγείας, έχουν επίσης συζητηθεί. Οι πρόσφατες ανακαλύψεις στη νανοτεχνολογία έχουν τη δυνατότητα να επιφέρουν μια αλλαγή παραδείγματος στην οδοντιατρική [Neel et al., 2015]. Ενώ η συντήρηση της στοματικής υγείας αποτελεί μεγάλη πρόκληση, διάφορα υλικά έχουν χρησιμοποιηθεί για τη θεραπεία διαφόρων οδοντιατρικών προβλημάτων αλλά η επιτυχία της θεραπείας περιορίζεται από τα βιοϋλικά που χρησιμοποιούνται. Υλικά που περιέχουν νανοσωματίδια (NPs) μπορούν να χρησιμοποιηθούν σε οδοντιατρικές εφαρμογές όπως στην ενδοδοντία, την περιοδοντολογία, την ορθοδοντική, τη στοματική χειρουργική, τη μηχανική των ιστών και σε άλλες, ώστε αυτοί οι περιορισμοί να ξεπεραστούν [Bapat et al., 2018]. Πολλές εφαρμογές της νανοτεχνολογίας στην οδοντιατρική είναι ακόμα στα αρχικά τους στάδια. Ένας αυξανόμενος αριθμός από προϊόντα μελετώνται επί του παρόντος, ενώ ορισμένα είναι πλέον εμπορικά διαθέσιμα [Mantri et al., 2021]. Η νανοτεχνολογία αναμένεται να έχει σημαντικό αντίκτυπο στην οδοντιατρική έρευνα και τις θεραπευτικές προσεγγίσεις της στο εγγύς μέλλον, έχοντας ως αποτέλεσμα την ενίσχυση της φροντίδας της στοματικής υγείας [Sharan et al., 2017].Abstract Nanotechnology has rapidly spread into all fields of science, providing significant alternative approaches to scientific and medical questions and problems. Nanotechnology has also had a huge impact on the health-care industry, and its use is a benefit not only to modern medicine but also dentistry. It is expected to pervade and further redefine dentistry, with potential applications encompassing all facets of oral illnesses, diagnosis, prevention, and treatment. Materials’ science in dentistry has embraced technology to create nanomaterials that are used in a variety of dental applications. These emerging technologies have the potential to provide significant benefits in the form of advancements in dental science and society [Sharan et al., 2017]. Manufacturers have included a variety of nanoparticles into dental materials in order to improve the chemical and physical properties of these materials. This field of research has been entitled ‘nanodentistry’ [Padovani et al., 2015]. Nanotechnology has been used in the development of restorative materials in dentistry with considerable success. The recent advances in nanodentistry and innovations in oral health-related diagnostic, preventive, and therapeutic methods required to maintain and obtain perfect oral health, have also been discussed. Recent breakthroughs in nanotechnology have the potential to bring about a paradigm shift in dentistry [Neel et al., 2015]. While oral health maintenance is a major challenge, various materials have been utilized to treat various dental problems, but the success of treatment is restricted by the biomaterials used. Materials including nanoparticles (NPs) can be employed in dental applications such as endodontics, periodontics, orthodontics, oral surgery, tissue engineering, and more to overcome these restrictions [Bapat et al., 2018]. Many applications of nanotechnology in dentistry are still in their early stages. A growing number of products are currently being studied, while some are commercially available [Mantri et al., 2021]. Nanotechnology is expected to have a significant impact on dental research and treatment approaches in the near future, resulting in enhanced oral health care [Sharan et al., 2017]

NEW DIMENSIONS INTO PROTEIN-NUCLEIC ACIDS INTERACTIONS

In the late 19th century, scientists microscopically observed the association of proteins with DNA strands. Since then, researchers have used a variety of in vitro and in vivo assays to demonstrate that proteins interact with DNA and RNA to influence the structure and function of the corresponding nucleic acid. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination and RNA processing and translocation continues to revolutionize our understanding of cell biology, normal cell development and the mechanisms of disease. Furthermore, there is the potential for constructing new molecules with excellent functionalities by assembling the very simple elements and components that are the functional subunits of these natural biopolymers. In this work of thesis we attempted making a new step forward a better knowledge of protein-nucleic acid biopolymers, focusing on two complexes of strong interest. One of the important factors for determining the expression of the function is the structure of the biopolymer. Clarification of the relationship between the structure and the function of biopolymer is of crucial importance to the development of methods for constructing tailor-made functional molecules. The first goal of my thesis focalized on the obtainment of a structural model of a well-known protein-DNA complex: Maf DNA binding domain and it DNA target (T-MARE was used in this study) with the aim of outlining a strategy for the obtainment of activity modulators. Both in silico simulations and experimental studies were carried out leading to the definition of a modus-operandi based on a disorder-order transition of the complex which is commendatory for the protein activation. On the other hand, a number of artificial enzymes have been recently constructed by using the molecular design based on structural information, screening methods that utilize libraries or by a combination of these two methods. However, the above mentioned approaches use single proteins or single nucleic acid as the structural unit, and the activity of the constructed artificial enzymes is remarkably lower in many cases, than that of the native enzymes. The construction of functional complexes (rather than a single biopolymer) as scaffold can be considered as one potential solution to these drawbacks. Further in my studies, using the HIV-Rev peptide and RRE (Rev Responsive Element) RNA complex as a scaffold, for which the tridimensional structure was fully characterized, the assemble of ribonucleopeptidic fluorescent sensors was accomplished in a stepwise manner. In this method, a randomized nucleotide sequence was introduced into the RNA subunit of RNP to construct a RNP library on which the in vitro selection method was applied. In the second step, the Rev peptide was modified with the fluorophore without altering the affinity and specificity of the RNP receptor. In the absence of a ligand for RNP, fluorescence emission was effectively quenched in the RNP complex, but recovered upon ligand binding. RNP sensors were thus created, as the ligand-binding event can be monitored by measurement of the fluorescence signals

Development of microRNA triggered therapeutic oligonucleotides and gold nanoparticle conjugates to improve specificity of RNA therapeutics

RNA-targeting oligonucleotide therapeutics and their nanoparticle conjugates hold great promise in treating intractable diseases, but their clinical applications are still limited by significant barriers including the lack of tissue or cell type specificity. Current strategy to improve tissue or cell type specificity of oligonucleotides therapeutics mainly involves conjugation with ligands. However, this strategy encounters bottleneck in diseased conditions where a specific surface marker is absent. In addition to protein markers, transcriptomic techniques have revealed complex and diverse alterations of coding and non-coding transcripts in different tissues, cell types or disease conditions, which opens up opportunities to control the activity of oligonucleotide therapeutics utilizing these endogenous transcripts to improve their specificity. The overall hypothesis of the dissertation is that using specific transcripts as triggering stimulus, oligonucleotides and their nanoparticle conjugates can be activated via toehold-mediated strand displacement reaction to conditionally regulate gene expression. As a proof-of-concept, we chose miRNA as the transcript trigger, hoping to provide a foundation for future design of smart therapeutics sensing more complicated transcript inputs. In this dissertation, we demonstrated the idea of miRNA-inducible conditional gene regulation agents with two models: (1) miR-33 triggered activation of DNAzyme-gold nanoparticle (AuNP) conjugates to down regulate tumor necrosis factor α (TNFα) in pro-inflammatory macrophages; and (2) miR-122-indicible antisense to down regulate hypoxia inducible factor 1α (HIF1α) in liver cells. In addition, to gain insights on the intracellular fate of oligonucleotide-AuNP conjugates for better design of conditional gene regulatory agents, we leveraged a powerful imaging modality, fluorescence lifetime imaging (FLIM), to monitor the intracellular integrity of oligonucleotide-AuNP conjugates. Programmable therapeutics with controllability of location, timing and intensity of their activity can lead to precise medicine with minimal side effects. We envision that the design principles for conditional oligonucleotides and their AuNP conjugates discovered from this dissertation could be adopted to a variety of translatable clinical applications and improve the controllability and safety of oligonucleotide therapeutics and nanoparticle conjugates.Ph.D

The Boston University Photonics Center annual report 2012-2013

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2012-2013 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This report summarizes activities of the Boston University Photonics Center during the period July 2012 through June 2013. These activities span the Center’s complementary missions in education, research, technology development, and commercialization. The Photonics Center continues to grow as an international leader in photonics research, while executing the Center’s strategic plan and serving as a university-wide resource for several affiliate Centers. For more information about the strategic plan, read the Photonics Center Strategic Plan section on page 10. In research, Photonics Center faculty published nearly 150 journal papers spanning the field of photonics. A number of awards for outstanding achievement in education and research were presented to Photonics Center faculty members, including a Peter Paul Professorship for Professor Xue Han, an NSF Career Award for Professor Ajay Joshi, and the 2012 Innovator of the Year Award from Boston University for Professor Theodore Moustakas. New external grant funding for the 2012- 2013 fiscal year totaled over $21.8M. For more information on our research activities, read the Research section on page 24. In technology development, the Photonics Center has turned a chapter, by completing the transition from a focus on Defense/ Security applications to a focus on the healthcare market sector. The commercial sector is expected to energize the technology development efforts for the foreseeable future, but the roots in defense/security are still important and the Center will continue to pursue new research grants in this area. For more information on our technology development program and on specific projects, read the Technology Development section on page 45. In education, 20 Photonics Center graduate students received Ph.D. diplomas. Photonics Center faculty taught 32 photonics courses. The Center supported a Research Experiences for Teachers (RET) site in Biophotonic Sensors and Systems for 10 middle school and high school teachers. The Photonics Center sponsored the Herbert J. Berman “Future of Light” Prize at the University’s Scholars Day. For more on our education programs, read the Education section on page 54. In commercialization, Boston University’s Business Innovation Center (BIC) currently hosts seven technology start-up companies. There is a healthy turnover in the Innovation Center space with a total of 19 companies residing at BIC over the past year. The mix of companies includes: life sciences, biotechnology, medical devices, photonics, and clean energy; and nine of the 19 companies originated from within BU. All the BIC tenants are engaged in the commercialization of new technologies of importance to society and all are active in the BU community in terms of offering internships, employment opportunities or research collaborations. For more information about Business Innovation Center activities, read the Business Innovation Center chapter in the Facilities and Equipment section on page 66