Click chemistry for the modification of oligonucleotides and their applications

This PhD thesis reports the published work done in the laboratories of baselick GmbH and the Ludwig Maximilians Universität (LMU). baseclick GmbH was founded by Prof. Dr. Thomas Carell (LMU) in collaboration with the chemical company BASF in 2008. Main focus of baseclick is the usage of click chemistry for modification of nucleic acids. In the described research work click chemistry is applied to oligonucleotides and in particular to DNA nanostructures, to mRNA to be then used in drug development and to produce highly labeled probes for fluorescent in situ hybridization. Continuing the previous work done at baseclick, at first click chemistry was applied on DNA nanostructures. In this field DNA, is not used as a carrier of genetic information but as material for the production of structures with different size, geometry and shape. The main concept behind the technology is based on Watson and Crick base pairing interactions, which bring portions of a ssDNA to hybridize with a complementary sequence, usually of another ssDNA strand, to form a rigid dsDNA helix. This is used in the rational design of the sequences to form complex structures with nanometric precision. DNA nanostructures and click chemistry were used to find an alternative to the current state of the art methods for gene synthesis in vitro. To date enzymatic synthesis of long DNA fragment is the method of choice since solid phase synthesis is not suitable for very long sequences. Enzymatic synthesis approaches are based on the activity of either DNA polymerases or DNA ligases reactions, but those methods suffer from some limitations: e.g. in the case of ligases the final gene is assembled by overlapping of strands then ligated to form a longer fragment, but it starts to be insufficient when a big number of strands need to be ligated together. With DNA polymerases the final product is formed by different cycles of the enzyme in a multiple step assembly, with the limitation due to the mispriming and formation of secondary structures which then lead to errors. Therefore in here, in collaboration with the group of Prof. Dr. Tom Brown from University of Oxford, a method was developed where, with the help of the DNA origami technique, ligase activity is replaced by chemical ligation, in this case click chemistry. 14 oligonucleotides were designed and synthesized with a 5’-terminal azide and 3’-terminal alkyne. The oligos where then preorganized in a DNA nanostructure to bring the alkyne and azide in close proximity and, most importantly, in a selective order. In this way after the click reaction occurs, the full length of the defined sequence is established, with a bio-compatible triazole linkage replacing the phosphate bond at the point of the oligo connections. In a second project click chemistry was used to stabilize a DNA nanostructure, in this case composed of 24 different interlocked oligonucleotides, and at the same time achieve selective labeling with biotin molecules in a one pot reaction. This work was done in collaboration with the group of Prof. Dr. Silvia Biocca of University of Rome Tor Vergata. DNA nanostructures thanks to their properties such as bio-compatibility, non-toxicity and bio-degradability have been used for different applications: e.g. drug delivery, nanocontainer, cellular biosensor and in vivo imaging. Anyhow, crucial for these applications is the understanding of how different DNA nanostructure enter the mammalian cells. For this reason, in this work five different topological configurations and functionalizations, with size varying from 8 to 80 nm and shape from tetrahedral, octahedral, cylindrical, square box and rectangular, have been investigated for their ability to interact with the scavenger receptor LOX-1, which overexpression has been associated with tumor development in many cancer cells. Inspired by the big success that mRNA therapy has in the last decade, methods to enable modification of very long RNA oligonucleotides, such as mRNA, were established using click chemistry. In vitro transcribed (IVT) mRNA, consisting in mRNA produced by RNA polymerases from a DNA template, is nowadays considered to be a valid candidate for a novel class of drugs. It was already demonstrated to be efficient in several diseases including: vaccination, protein replacement and cancer therapy. Indeed nowadays mRNA is playing a central role in vaccination programs against SARS-Cov-2 pandemic. The main idea behind the mRNA therapy is to provide IVT mRNA to the patients to help them developing their own cure. For example in vaccination, mRNA coding for a specific viral antigen is used to produce an immune response leading to the immunogenicity. Besides stability issues deriving from using RNA molecule as drugs, another problem arises from the cellular delivery of such molecules. Indeed, delivery to a specific target is still an unsolved problem. To date the mRNA is delivered to patients using lipid nanoparticle (LNPs) to act as carrier and at the same time as a barrier from the extracellular environment. Recent studies demonstrate however that the usage of LNP is not the ideal method to deliver mRNA. It has been proven to be less efficient in vivo than what was observed in vitro. Especially in living organisms the main destination is the liver, which is often not the final target for the therapy. Also driven by the recent FDA approval of the first siRNA drug (GIVLAARITM), where the siRNA has been chemically modified using N-acetylgalactosamine molecules (GalNac) to enable efficient targeted delivery, it is described here a chemoenzymatic approach based on the incorporation of modified nucleotides bearing an alkyne or azide moiety (such as 5-ethynyl-UTP or 3’-azido-dd-ATP), that can then be labelled post-transcriptionally, using click chemistry. This method allows for example the incorporation of specific modifications inside the mRNA that would not be accepted by the RNA polymerases, e.g. targeting-molecules for specific delivery, or fluorescent dyes for tracking, thus potentially improving the biochemical properties of the mRNA. Click chemistry was also used to improve the current methods for the preparation of probes used in fluorescent in situ hybridization (FISH). FISH is a cytogenetic analysis that allows the detection and the spatial localization of specific nucleotide sequences in tissues or cells. The fluorescent probes, consisting of ssDNA, were designed to hybridize only to those parts of the target DNA/RNA with a high degree of complementarity. Then by utilizing fluorescent microscopy it was possible to localize where the fluorescent probes are hybridized. This technique is largely used for diagnosis of genetic abnormalities such as gene fusion, aneuploidy, loss of chromosomal regions, detection of oncogenes and diagnosis of viral infections and to date it can also detect other targets such RNA (mRNA) in cells and tissue samples. The probes can be prepared in various ways, such as nick translation, random priming, PCR, end labelling or NHS-ester chemistry. Most of the probes, especially for RNA detection, are composed of ssDNA which are approximately 20-25 nt long, conjugated to a fluorophore via coupling of an amino group introduced at the 3’ end and an activated ester of the fluorophore. Finally a method for preparation of mRNA FISH probes based on click chemistry is described, where each probe contains three fluorophore instead of a single one, thus giving an increment of fluorescent yields and allowing microscopy analysis without the need of special deconvolution software. Furthermore this allows the detection using fluorescent activated cell sorting (FACS) devices. Enabling FACS analysis is of outmost importance especially for clinical studies, where up to now the detection of specific mRNA or chromosomes sequences is still done manually by clinicians, analyzing all the samples through visual inspection

Development of a novel wearable system for real-time measurement of the inter-foot distance during gait

The combination of magneto-inertial measurement unit (MIMU) and distance sensor (DS) represents smart solution for evaluating the distance between feet during various daily-life activities. In particular, when analyzing gait, the latter technology can be used for estimating the instantaneous or average distance between selected points of the feet (IFD) during mid-swing and mid-stance phases. The aim of this preliminary work is twofold: a) to develop and validate a novel wearable system for the measurement of the IFD during gait; b) to investigate the optimal positioning of the DS on the foot. Preliminary results showed that the innovative wearable system can be effectively used for accurately measuring the IFD during gait. Interestingly, the accuracy of the IFD estimation is highly affected by the position of the DS on the foot

A wearable solution for accurate step detection based on the direct measurement of the inter-foot distance

Accurate step detection is crucial for the estimation of gait spatio-temporal parameters. Although several step detection methods based on the use of inertial measurement units (IMUs) have been successfully proposed, they may not perform adequately when the foot is dragged while walking, when walking aids are used, or when walking at low speed. The aim of this study was to test an original step-detection method, the inter-foot distance step counter (IFOD), based on the direct measurement of the distance between feet. Gait data were recorded using a wearable prototype system (SWING2DS), which integrates an IMU and two time-of-flight distance sensors (DSs). The system was attached to the medial side of the right foot with one DS positioned close to the forefoot (FOREDS) and the other close to the rearfoot (REARDS). Sixteen healthy adults were asked to walk over ground for two minutes along a loop, including both rectilinear and curvilinear portions, during two experimental sessions. The accuracy of the IFOD step counter was assessed using a stereo-photogrammetric system as gold standard. The best performance was obtained for REARDS with an accuracy higher than 99.8% for the instrumented foot step and 88.8% for the non-instrumented foot step during both rectilinear and curvilinear walks. Key features of the IFOD step counter are that it is possible to detect both right and left steps by instrumenting one foot only and that it does not rely on foot impact dynamics. The IFOD step counter can be combined with existing IMU-based methods for increasing step-detection accuracy

A proximity sensor for the measurement of the inter-foot distance in static and dynamic tasks

Measuring the base of support is of paramount importance in determining human stability during gait or balance tests. While wearable inertial sensors have been successfully employed to quantify numerous gait parameters (velocity, stride length, etc), they could not be used to estimate quantities related to the feet relative position. Thus, alternative technological solutions need to be investigated. Some attempts have been made by combining light intensity infrared or ultrasounds sensors with inertial measurement units. Lately, the Infrared Time-of-Flight technology (IR-ToF) has become popular for measuring distances. IR-ToF sensor measures the time an electromagnetic wave needs to travel a distance. The aim of this work was to investigate the feasibility of the use of an IR-ToF sensor for estimating the inter-foot distance (IFD) in both static and dynamic tasks. Very accurate IFD estimates were obtained during Static (MAE%=3.3%) and Oscillation (MAE%=4.1%) conditions, while larger errors during Gait trials (MAE%=19.8%)

Static and dynamic accuracy of an innovative miniaturized wearable platform for short range distance measurements for human movement applications

Magneto-inertial measurement units (MIMU) are a suitable solution to assess human motor performance both indoors and outdoors. However, relevant quantities such as step width and base of support, which play an important role in gait stability, cannot be directly measured using MIMU alone. To overcome this limitation, we developed a wearable platform specifically designed for human movement analysis applications, which integrates a MIMU and an Infrared Time-of-Flight proximity sensor (IR-ToF), allowing for the estimate of inter-object distance. We proposed a thorough testing protocol for evaluating the IR-ToF sensor performances under experimental conditions resembling those encountered during gait. In particular, we tested the sensor performance for different (i) target colors; (ii) sensor-target distances (up to 200 mm) and (iii) sensor-target angles of incidence (AoI) (up to 60°). Both static and dynamic conditions were analyzed. A pendulum, simulating the oscillation of a human leg, was used to generate highly repeatable oscillations with a maximum angular velocity of 6 rad/s. Results showed that the IR-ToF proximity sensor was not sensitive to variations of both distance and target color (except for black). Conversely, a relationship between error magnitude and AoI values was found. For AoI equal to 0°, the IR-ToF sensor performed equally well both in static and dynamic acquisitions with a distance mean absolute error <1.5 mm. Errors increased up to 3.6 mm (static) and 11.9 mm (dynamic) for AoI equal to ±30°, and up to 7.8 mm (static) and 25.6 mm (dynamic) for AoI equal to ±60°. In addition, the wearable platform was used during a preliminary experiment for the estimation of the inter-foot distance on a single healthy subject while walking. In conclusion, the combination of magneto-inertial unit and IR-ToF technology represents a valuable alternative solution in terms of accuracy, sampling frequency, dimension and power consumption, compared to existing technologies

Government policies to enhance access to credit for infrastructure-based PPPs:an approach to classification and appraisal

Analysis of ways the public sector can ease the use of private money for infrastructure financing

A Research Framework for the Multidisciplinary Design and Optimization of Wind Turbines

The design of very large wind turbines is a complex task which requires the development of dedicated tools and techniques. In this chapter, we present a system-level design procedure based on the combination of multi-body numerical models of the turbine and a multilevel optimization scheme. The overall design aims at the minimization of the cost of energy (COE) through the optimization of all the characteristics of the turbine, and the procedure automatically manages all the simulations required to compute relevant loads and displacements. This unique setup allows the designer to conduct trade-off studies in a highly realistic virtual environment and is an ideal test bench for advanced research studies in which it is important to assess the economic impact of specific design choices. Examples of such studies include the impact of stall-induced vibrations on fatigue, the development of active/passive control laws for large rotors, and the complete definition of 10–20 MW reference turbines

Benign paroxysmal positional vertigo following whiplash injury: a myth or a reality?

Objective: The aim of the study was to evaluate the true incidence, diagnosis, and treatment of benign paroxysmal positional vertigo (BPPV) arising after whiplash injury and to distinguish this type of posttraumatic vertigo from other types of dizziness complained after trauma. Methods: This was a retrospective study comprising patients referred to our center after whiplash injury. The patients were evaluated with neurotologic examination including bedside and instrumental tests. A Dizziness Handicap Inventory evaluating the symptoms of patients was submitted before and after treatment and was evaluated. The BPPV patients were separately evaluated from those with cervicogenic vertigo, and a comparison between our data about idiopathic BPPV was done. Results: Eighteen patients of whiplash who had BPPV were evaluated. The mean age was 38.2 years. BPPV was the cause of vertigo in 33.9% of total whiplash patients. In 16 cases, the posterior semicircular canal was involved; the lateral semicircular canal was involved in 2 cases. The instrumental neurotologic assessment did not show any alteration of either vestibulospinal reflexes or dynamic ocular movements. Duration of symptoms before treatment ranged from 3 to 26 days. A total of 55.5% of patients had relief from their symptoms after first repositioning maneuver. The Dizziness Handicap Inventory score improved in all patients treated with repositioning maneuvers, but no difference emerged with idiopathic BPPV data. Conclusion: BPPV after whiplash injury could be unveiled with a simple bedside examination of peripheral vestibular system, and a treatment could be done in the same session. The diagnosis of posttraumatic BPPV is not different from the idiopathic form, but the treatment may require more maneuvers to achieve satisfactory results