Распознавание нуклеиновых кислот с использованием многокомпонентных ДНК наноконструкций / Recognition of nucleic acids using multicomponent DNA nanoconstructs тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Эльдиб Ахмед Абделкадер Мохамед Отман

• Эльдиб Ахмед Абделкадер Мохамед Отман
• кандидат наук
• 2025

Оглавление диссертации кандидат наук Эльдиб Ахмед Абделкадер Мохамед Отман

TABLE OF CONTENTS

INTRODUCTION

CHAPTER 1. LITERATURE REVIEW

1.1. Cancer

1.1.1. Cancer markers

1.1.1.1. RNA-based Cancer markers

1.2. RNA containing viruses

1.3. Corona viruses

1.4. Gene therapy

1.5. Polymerase chain reaction (PCR)

1.5.1. Real time-PCR

1.6. DNAzymes

1.6.1. DNAzyme as a gene therapy tool

1.6.2. Binary (split) DNAzyme for nucleic acid detection

1.6.3. Binary DNAzyme in molecular computation

1.6.4. Binary DNAzymes-based DNA-machines

1.6.5. Antisense oligonucleotides

1.6.6. RNase H

1.6.7. Modified Antisense oligonucleotides

CHAPTER 2. MATERIALS AND METHODS

2.1. Reagents and equipment used

2.1. Fluorescence Spectrophotometry Test

2.2. Binary Threshold DNAzyme fluorescence assay

2.4. DNA logic gates/ thresholding machines cleavage assay

2.5. Viral Diagnostics DNA-machine fluorescence assay

2.5.1. Titration assay

2.5.2. Limit of detection

2.6. Detection of viral RNA in clinical samples assay

2.7. Detection of extracted viral RNA from cell culture and selectivity assay

CHAPTER 3. RESULTS AND DISCUSSION

3.1. Molecular Computation

3.1.1. Selection of hsa-miR-17-5p (miR-17) microRNA as input for DNA thresholding gate

3.1.3. Selection of DAD-1 mRNA (RNA-46) as target and substrate for DNAzyme thresholding gates and DNA machines

3.1.4. Threshold Split DNAzyme gates

3.1.4.1. Design of the DNAzyme thresholding gates

3.1.4.2. Optimization and activity assessment of the thresholding split DNAzyme gates targeting F_sub

3.1.4.3. Selectivity assay of the thresholding split DNAzyme gates

3.1.4.4. Cleavage assessment of the thresholding split DNAzyme gates targeting RNA-46

3.1.5. Thresholding DNA nanomachines (Th-DNM)

3.1.5.1. Design of the thresholding DNA nanomachines

3.1.5.2. Assembly of the thresholding DNA nanomachines

3.1.5.3. Cleavage efficiency assessment and selectivity testing of the thresholding DNA nanomachines targeting F_sub

3.1.5.4. Cleavage efficiency assessment and comparison of the thresholding DNA nanomachines targeting DAD-1 mRNA (RNA-46)

3.1.6. Selection of miR-92 as a cancer marker and input for Threshold Binary DNazyme

3.1.7. Selection of GFP_RNA-60 mRNA as RNA target for thresholding split DNAzyme gates

3.1.8. Threshold split DNAzyme gates

3.1.8.1. Design of thresholding gates

3.1.8.2. Optimization and activity assessment of the thresholding split DNAzyme gates targeting F_sub

3.1.8.3. Cleavage efficiency assessment and comparison of the thresholding split

DNAzyme gates targeting GFP-RNA

3.1.9. Selection of cancer markers as inputs for the activation of designed DNA logic gates

3.1.9.1. Selection of hsa-miR-15a-5p (miR-15; 1st input) and hsa-miR-21-5p (miR-21; 3rd input) as activating input signals

3.1.9.2. Selection of hsa-miR-7d-5p (miR-7; 4th input) as an inhibitory input signal

3.1.10. DNA logic gates

3.1.10.1. Design of the YES gate

3.1.10.2. Cleavage efficiency assessment of YES gate

3.1.10.3. Design of 3iAND and interchangeable 2iAND gates

3.1.10.4. Efficiency assessment of 2iAND and 3iAND gates

3.1.10.5. Design of 2iINHIBIT and 3iINHIBIT gates

3.1.10.6. Efficiency assessment of 2iINHIBIT and 3iINHIBIT gates

3.1.10.7. Adaptation of DNA logic gates for the cleavage of a synthetic mRNA fragment (GFP-60)

3.1.11. ASO-based DNA thresholder agents

3.1.11.1. Design of ASO-based DNA thresholder agents

3.1.11.2. Optimization and cleavage efficiency assessment of ASO-based DNA thresholders targeting selected GFP-RNA

3.1.11.3. Selectivity assay of ASO-based DNA thresholders

3.2. Diagnostics

3.2.1. SARS-CoV-2 sample collection, cell culture and isolation of virus

3.2.2. The 4-Arm DNA machine

3.2.2.1. Design of the 4-Arm DNA machine (4DNM)

3.2.2.2. Assembly of the 4-Arm DNA machine (4DNM)

3.2.2.3. Fluorescent assay of the 4DNM and the Bi-Dz targeting a synthetic DNA fragment of SARS-CoV-2

3.2.2.4. Sensitivity and selectivity assessment between the 4DNM and the binary

DNAzyme in detecting the viral particles obtained from the Vero CCL-81 cell line

3.2.2.5. Detection of viral particles in clinical samples

3.2.3. The hook-equipped DNA nanomachine (HDNM)

3.2.3.1. Design of the hook-equipped DNA nanomachine

3.2.3.2. Assembly of the hook-equipped DNA nanomachine

3.2.3.3. Fluorescent assay detecting a synthetic DNA fragment of SARS-CoV-2 RNA (COV2-DNA1)

3.2.3.4. Detection of SARS-CoV-2 RNA in total RNA isolated from infected Vero cells

3.2.3.5. Selectivity assay of the hook-equipped DNA nanomachine

3.2.4. The multicore DNA nanomachine (MDNM)

3.2.4.1. Design of the multicore DNA nanomachine

3.2.4.2. Assembly of the multicore DNA nanomachine

3.2.4.3. Fluorescent assay detecting a synthetic DNA fragment of SARS-CoV-2 RNA (COV2-DNA190)

3.2.4.4. Detection of SARS-CoV-2 RNA in total RNA isolated from infected Vero cells

3.2.4.5. Assessment of cooperativity of all four cores in MDNM

CONCLUSION

LIST OF ABBREVIATIONS AND CONVENTIONS

REFERENCES

Введение диссертации (часть автореферата) на тему «Распознавание нуклеиновых кислот с использованием многокомпонентных ДНК наноконструкций / Recognition of nucleic acids using multicomponent DNA nanoconstructs»

INTRODUCTION

Actuality of the research topic. Hybridization of nucleic acid probes to RNA is used in diagnosis and therapy. This approach leverages the intrinsic properties of nucleic acids to recognize and bind to specific sequences, enabling the development of targeted therapeutic and specific and sensitive diagnostic tools. This work applies the approaches of DNA nanotechnology in the design of a new generation of hybridization probes for applications in the two key areas: treating and diagnosing human diseases.

DNA hybridization probes have emerged as powerful tools for both diagnostics and therapy, leveraging their ability to selectively bind to specific nucleic acid sequences [88]. These probes offer high specificity and can be designed to recognize genetic disease markers, making them invaluable in analysis and therapy of human diseases [168]. It was demonstrated earlier that using multiple hybridization probes (elements) can improve selectivity, sensitivity and multiplexing capability of hybridization-based approaches [83]. These functional elements can be combined in a single nanostructure called 'DNA Constructs', with improved cooperativity in recognition of DNA and RNA [105]. It was hypothesized that hybridization probes and DNA Constructs are evolving in DNA nanorobots, possessing the following four functions: (i) sensing for recognition of disease markers, (ii) computation for analysis multiple markers, (iii) actuation that can accomplish therapeutic treatment, and (iv) auto delivery [68]. This study is devoted to advancing sensing and computation functions of the DNA nanorobot.

The study takes advantage of the earlier developed hybridization sensor based on RNA-cleaving DNAzyme (Dz). The sensor, named 'binary Dz' or biDz can bind specific nucleic sequences followed by activation of RNA cleaving function, which can be translated in a fluorescent signal for sensing. The biDz sensor can be equipped with multiple recognition sites for binding several different nucleic acid analytes. The analyte is a disease marker that will activate the DNA construct once it is detected. In this work, DNA Constructs based on biDz was adopted to (i) differentiation low from high concentrations of cancer marker RNA sequences; (ii) recognition of multiple biomarker sequences; (iii) detecting low concentrations of viral RNA in amplification free format.

Degree of development of the research topic. Research in the development of DNA nanorobots with integrated sensing and computational modules aimed at disease diagnostics and therapy is currently in an active stage of advancement. In recent years, significant attention has been directed toward the creation of DNA structures capable of performing complex logical operations at the molecular level, opening new horizons for the application of DNA nanotechnology in biomedicine. Research in DNA nanotechnology is also focused on the development of oligonucleotides that, once they bind to the target, trigger RNA cleavage, such as antisense oligonucleotides (ASOs) [55] or RNA-cleaving DNAzymes. However, techniques that lack input activation-based control and target selection, since they mainly target oncogenes and do not kill the cancer cell directly, still face the problem of being activated in healthy cells, which can lead to deadly side effects.

DNA nanomachines capable of simultaneously performing multiple tasks, including detection and RNA cleavage, such as those developed by [158], address the activation control problem and suggest targeting housekeeping gbaenes to cause toxicity in unhealthy cells. However, the detection of biomarkers, especially folded RNA molecules, can be a significant challenge. Moreover, detecting one input may be useful in the case of viral diseases, but in other complex diseases, such as cancer, multiple markers are needed to determine whether a cell is healthy. Thus, a critical task is the development and optimization of algorithms for signal recognition and processing.

DNA logic gates proposed by Khanum et al. (2020), including YES, 2I AND, and 3I AND gates based on DNAzymes, lack the ability to connect the logic gate elements within one structure, which remains an obstacle in developing a DNA processor to analyze the algorithms. Furthermore, there is a need to design DNA logic gates that activate or inhibit based on cancer-related markers.

Despite significant progress in this area, questions remain regarding the integration of various modules into a single system, the sensitivity of constructs, and the minimization of side effects associated with their use in the body. Active research is also ongoing on the development of new mechanisms for the regulation and activation of DNA

nanorobots, including the use of antisense and DNAzyme strategies and new types of logic elements.

Aim and Objectives of defense:

This study aims to create a foundation for using sophisticated DNA Constructs in diagnostic and therapy of human diseases. To achieve these aims, the following tasks were pursued:

1. Develop DNA constructs based on binary DNAzymes (biDz) and antisense oligonucleotides (ASO) to achieve in vitro cleavage of RNA targets in the presence of low and high concentrations of oncogenic microRNAs.

2. Develop DNA constructs based on biDz, operating on the principle of logic gates and incorporating a universal DNA scaffold that unites all functional components of the DNA constructs into a single complex, to achieve inhibition or activation of substrate cleavage in the presence of various combinations of oncogenic or tumor-suppressive microRNAs.

3. Develop DNA constructs based on biDz to achieve a reduction in the limit of detection (LOD) of analytes by adding to the DNA constructs: a) additional analyte-binding sites; b) a fluorogenic substrate delivery function; c) an increased number of catalytic centers.

Scientific novelty of the work. For the first time, cleavage of specific RNA target was achieved in the presence of high but not low concentrations of as miR-17 and miR-92. The threshold concentration that triggers construct activating can be fine-tuned by changing the number of recognition modules in DNA Constructs. For the first time, DNA logic gates with YES, 2AND, 3AND, 2iINHIBIT, and 3iINHIBIT Boolean logic functions were integrated to a single DNA scaffold. These gates enabled RNA cleavage through different combinations of miR-15, miR-17, and miR-21, with specific signal-to-background ratios. For the 1st time it was demonstrated that addition of RNA binding arms, Substrate delivery and additional substrate cleaving functions to DNA Construct significantly reduces LOD of viral RNA extracted from infected cells.

Value of scientific work. The results advance the field of DNA nanotechnology toward application in diagnosis and treatment of human diseases. The developed DNA -constructs recognize concentrations and patterns of nucleic acid analytes autonomously without involvement of a human operator, which create a basis for development of DNA nanorobot by analysis for distinguishing healthy from non-healthy cells. The PCR-free detection of low RNA concentration creates a bases for the development of highly sensitive sensing modules of DNA nanorobots and could be used in PCR-free diagnostics of human infectious diseases.

Research methodology and methods. The chosen RNA targets included fragments of GFP RNA (RNA-60) and DAD-1 RNA (RNA-46), for the convent monitoring of gene suppression in cell culture experiments, and viral RNA SARS-CoV-2, due to its significant global health threat posed by high mutation rates of RNA viruses. Specific miRNAs, such as miR-15-5p, miR-17-5p, miR-21-5p, miR-7d-let, miR-92a-1 (miR-15, miR-17, miR-21, miR-7, miR-92), were selected as cancer markers because of their elevated expression levels in various cancers, providing a relevant and challenging target for DNA-based probes. The main research methods included polyacrylamide gel electrophoresis (PAGE) and fluorescence-based quantitative analysis of RNA cleavage products and statistical analysis of the results.

Statements of defense:

1. In vitro RNA cleavage can be achieved only at high but not low concentrations of cancer markers by adding more marker binding sites to the biDz-based DNA Constructs.

2. In vitro RNA cleavage can be achieved only at high but not low concentrations of cancer markers by adding more marker binding sites to the ASO-based DNA Constructs.

3. Logic gate DNA Constructs containing all functional elements as part of a single DNA association can cleave target RNA only in the presence of a selected set of microRNA cancer markers that corresponds to behaviour of AND and INHIBIT Boolean logic operations.

4. The addition of RNA binding arms to biDz-based DNA Constructs decreases limit of detection (LOD) for detection of viral DNA and RNA analytes folded in stable secondary structures.

5. Addition fluorogenic substrate delivery function to biDz-based DNA Constructs decreases the limit of detection (LOD) for detection of viral DNA and RNA analytes.

6. BiDz-based DNA Construct equipped with multiple RNA cleaving cores demonstrates less LOD for detection of viral DNA and RNA analytes in comparison with biDz sensor having single catalytic core.

Credibility of scientific achievements. To ensure and confirm the high degree of reliability in the work used a set of modern experimental methods of research, statistics. Scientific provisions, conclusions and recommendations are based on the analysis of a huge amount of scientific literature and confirmed by experimental data.

Research approbation. The results of the work are presented in 18 publications (articles, abstracts) - 7 of articles are included in the Scopus and Web of Science citation bases, 11 theses presented at 9 All-Russian and international conferences.

Заключение диссертации по теме «Другие cпециальности», Эльдиб Ахмед Абделкадер Мохамед Отман

CONCLUSION

This work demonstrated that complex multicomponent DNA Constructs can effectively recognize biologically significant RNA, laying the groundwork for applying DNA nanotechnology and molecular DNA nanorobots in disease detection and therapy.

1. 21 DNA constructs based on binary DNAzymes (biDz) and 3 DNA constructs based on antisense oligonucleotides (ASO) were developed. The DNA constructs BiRDz, 2i-DTh, 3i-DTh, 4i-DTh, and 5i-DTh activated RNA target cleavage at miR-92 concentrations of 0.25, 20, 80, 90, and 190 nM, respectively. DNA constructs containing DNA scaffold, such as DNM, Th-DNM-2i-46, and Th-DNM-3i-46, were activated at 2, 25, and 55 nM miR-17, respectively. The DNA constructs BiASO, 2i-A Th, and 3i-A Th were activated at miR-17 concentrations of 2.6, 7.5, and 39.5 nM, respectively. The threshold concentration of oncogenic microRNAs for DNA construct activation depended on the number of microRNA-binding sites in the DNA constructs.

2. 12 DNA constructs based on biDz were developed, containing all functional oligonucleotides attached to a single scaffold and operating on the principle of logic gates. The DNA constructs YES, 2iAND-A, and 2iAND-B demonstrated RNA target cleavage with signal-to-background ratios of 80.4, 10.3, and 39.2, respectively, after 7 hours of incubation. The DNA constructs 3iAND, 2iINHIBIT, and 3iINHIBIT demonstrated RNA target cleavage with signal-to-background ratios of 18.3, 4.3, 3.9, and 26, respectively, after 24 hours of incubation. The DNA constructs 2iINHIBIT and 3iINHIBIT showed complete inhibition of RNA target cleavage after the addition of the tumor suppressor miR-7 at a concentration 1 and 2 times higher than that of the DNA construct itself, respectively.

3. 3 DNA constructs were developed for the detection of DNA and RNA analytes of SARS-CoV-2. The 4DNM DNA construct, equipped with two additional RNA-binding sites, demonstrated a limit of detection (LOD) of 1 pM for the synthetic DNA analyte. The LOD for the RNA analyte was 26 pM after 3 hours of incubation, whereas biDz was unable to detect RNA. The HDNM DNA construct, with a substrate delivery function, showed an LOD of 0.063 pM and 0.025 pM after 1 and 3 hours,

respectively. The LOD for the RNA analyte was 26 pM and 2 pM after 1 and 3 hours, respectively. The MDNM DNA construct, with four catalytic centers, demonstrated an LOD for the synthetic DNA analyte of 6.5, 2.6, and 0.3 pM after 0.5, 1, and 3 hours, respectively. The LOD for the RNA analyte was 28 pM and 5 pM after 1 and 3 hours, respectively, showing improved sensitivity, as 4DNM did not detect viral RNA after 1 hour of incubation.

Recommendations and Prospects for Further Development. The results of this dissertation research can be utilized in the development of diagnostic and therapeutic approaches for oncological and viral diseases, as well as in expanding the application of DNA probes in biomedicine. Further modifications of DNA constructs may be conducted to lower the sensitivity threshold and enhance the efficiency of RNA target cleavage. Additionally, investigating the effects of various chemical modifications on the stability and catalytic activity of the constructs remains an important direction. Another promising avenue is the study of the kinetics of interactions between logical DNA constructs and microRNAs to predict response times. Adapting these technologies to modern molecular diagnostic platforms, miniaturizing DNA constructs, and integrating them into portable devices for rapid analysis are also key areas for future development. Moreover, continued research on biocompatibility and the development of safe methods for introducing DNA constructs into the body for potential therapeutic applications are necessary.

Список литературы диссертационного исследования кандидат наук Эльдиб Ахмед Абделкадер Мохамед Отман, 2025 год

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