Applications In Molecular Biology Research Articles

Design of ancestral mammalian alkaline phosphatase bearing high stability and productivity.

Sequence-based protein redesign methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR), have the potential to construct new enzymes utilizing protein sequence data registered in a rapidly expanding sequence database. The high thermostability of these enzymes would expand their application in diagnostics and molecular biology. These enzymes have also demonstrated a high level of active expression by Escherichia coli. These characteristics make these APs attractive candidates for industrial application. In addition, different amino acid residues are primarily responsible for thermal stability and active expression, suggesting important implications for strategies for designing enzymes by FCD and ASR.

USO DA BIOLOGIA MOLECULAR NA PRODUÇÃO DE NOVOS MEDICAMENTOS

INTRODUCTION: Molecular biology, by acting in the development of biotechnological drugs, such as Zidovudine and Sofosbuvir, offers significant innovations in modern pharmacology, providing more specific and effective therapies for complex diseases. These drugs, although promising, still face challenges related to high costs and limited accessibility. The field is constantly evolving, seeking to improve the specificity and sensitivity of therapies for better prognoses. GENERAL OBJECTIVE: To explore the applications of molecular biology in the production of new drugs and its impact on the creation of personalized treatments. METHODOLOGY: This work is a qualitative, exploratory bibliographic review, which analyzes 7 articles published between 2020 and 2024 on molecular biology correlated to drug development. The research used databases such as BVS, DrugBank, PubMed and others, applying rigorous criteria for their selection. RESULTS: This study analyzes the potential of molecular biology for the development of new drugs, highlighting techniques such as PCR, DNA sequencing and CRISPR/Cas9. A review highlights the importance of these approaches in personalizing therapies and identifying molecular targets, in addition to discussing the pharmacodynamics and pharmacokinetics of drugs. Advances in molecular pharmacology offer new opportunities for more targeted and specific treatments with several drugs in complex diseases. CONCLUSION: The integrative review highlighted the importance of molecular biology techniques, such as DNA sequencing, PCR and CRISPR/Cas9, in drug development and personalized medicine, demonstrating their effectiveness in the treatment of genetic and infectious diseases. It is urgent to carry out new studies to explore the interactions of these technologies and their clinical applications, evolving towards better pharmacological treatments.

Insights into phylogenetic relationships and gene rearrangements: complete mitogenomes of two sympatric species in the genus Rana (Anura, Ranidae).

Mitochondrial genomes (also known as mitogenomes) serve as valuable molecular markers and have found widespread applications in molecular biology and ecology. There is abundant sequence variation in vertebrate mitogenomes, and occasionally, they exhibit gene rearrangements. In this study, two Chinese endemic Rana species, Ranajiemuxiensis and Ranahanluica, were sequenced and analyzed to obtain their complete mitogenomes. The two species were sympatrically distributed in the Zhangjiajie National Forest Park, in Wulingyuan District, Zhangjiajie City, Hunan Province, China. The mitogenome of R.jiemuxiensis was 17,506 bp, while that of R.hanluica was 17,505 bp, each comprising 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a non-coding control region (D-loop). The gene content, nucleotide composition, and evolutionary rates of each mitogenome were analyzed and compared with those of congeners. A phylogenetic analysis based on 22 mitogenomes in Rana revealed that the two sympatric species were in two different lineages, indicating that they were genetically separated to a certain extent. Three types of gene rearrangement patterns were identified when examining the gene orders of the 22 Rana mitogenomes. Most of the species shared a second and dominant gene rearrangement pattern that originated from the first ancient pattern. A "tandem duplication - multiple deletion" hypothesis was proposed to explain the evolution of these different gene rearrangement patterns. This study provided valuable data references and enhanced our understanding of the phylogenetic implications and gene rearrangements of Rana species.

Intelligent guide RNA: dual toehold switches for modulating luciferase in the presence of trigger RNA

The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA—a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs—is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform.

More quantum chemistry with fewer qubits

Quantum computation is one of the most promising new paradigms for the simulation of physical systems composed of electrons and atomic nuclei, with applications in chemistry, solid-state physics, materials science, and molecular biology. This requires a truncated representation of the electronic structure Hamiltonian using a finite number of orbitals. While it is, in principle, obvious how to improve on the representation by including more orbitals, this is usually unfeasible in practice (e.g., because of the limited number of qubits available) and severely compromises the accuracy of the obtained results. Here, we propose a quantum algorithm that improves on the representation of the physical problem by virtue of second-order perturbation theory. In particular, our quantum algorithm evaluates the second-order energy correction through a series of time-evolution steps under the unperturbed Hamiltonian. An important application is to go beyond the active-space approximation, allowing to include corrections of virtual orbitals, known as multireference perturbation theory. Here, we exploit the fact that the unperturbed Hamiltonian is diagonal for virtual orbitals and show that the number of qubits is independent of the number of virtual orbitals. This gives rise to more accurate energy estimates without increasing the number of qubits. Moreover, we demonstrate numerically for realistic chemical systems that the total runtime has highly favorable scaling in the number of virtual orbitals compared to previous studies. Numerical calculations confirm the necessity of the multireference perturbation theory energy corrections to reach accurate ground-state energy estimates. Our perturbation theory quantum algorithm can also be applied to symmetry-adapted perturbation theory. As such, we demonstrate that perturbation theory can help to reduce the quantum hardware requirements for quantum chemistry. Published by the American Physical Society 2024

Leveraging analog quantum computing with neutral atoms for solvent configuration prediction in drug discovery

We introduce an approach to sampling equilibrium solvent water molecule configurations within proteins that leverages analog quantum computing. We present a complete end-to-end study from the molecular biology application to the development of the quantum algorithm to the implementation on a neutral atom quantum processing unit (QPU). To do so, we combine a quantum placement strategy to the 3D Reference Interaction Site Model, an approach capable of predicting continuous solvent distributions. The intrinsic quantum nature of such coupling guarantees molecules not to be placed too close to each other, a constraint usually imposed by hand in classical approaches. We present first a full quantum adiabatic evolution model that uses a local Rydberg Hamiltonian to cast the general problem into an antiferromagnetic Ising model. Its solution is embodied into a Rydberg atom array QPU. Following a classical emulator implementation, a QPU portage allows to experimentally validate the algorithm performances on an actual quantum computer. As a perspective of use on next generation devices, we emulate a second hybrid quantum-classical version of the algorithm. Such a variational quantum approach uses a classical Bayesian minimization routine to find the optimal laser parameters. Overall, these Quantum-3D-RISM algorithms open a route towards the application of analog quantum computing in molecular modeling and drug design. Published by the American Physical Society 2024

Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Improving long-term kidney allograft survival by rethinking HLA compatibility: from molecular matching to non-HLA genes.

Optimizing immunologic compatibility in organ transplantation extends beyond the conventional approach of Human Leukocyte Antigen (HLA) antigen matching, which exhibits significant limitations. A broader comprehension of the roles of classical and non-classical HLA genes in transplantation is imperative for enhancing long-term graft survival. High-resolution molecular HLA genotyping, despite its inherent challenges, has emerged as the cornerstone for precise patient-donor compatibility assessment. Leveraging understanding of eplet biology and indirect immune activation, eplet mismatch calculators and the PIRCHE-II algorithm surpass traditional methods in predicting allograft rejection. Understanding minor histocompatibility antigens may also present an opportunity to personalize the compatibility process. While the application of molecular matching in deceased donor organ allocation presents multiple technical, logistical, and conceptual barriers, rendering it premature for mainstream use, several other areas of donor-recipient matching and post-transplant management are ready to incorporate molecular matching. Provision of molecular mismatch scores to physicians during potential organ offer evaluations could potentially amplify long-term outcomes. The implementation of molecular matching in living organ donation and kidney paired exchange programs is similarly viable. This article will explore the current understanding of immunologic matching in transplantation and the potential applications of epitope and non-epitope molecular biology and genetics in clinical transplantation.

DNARS: A safe, environmentally friendly and high-quality DNA extraction method suitable for various biological samples

The functional and interactional relationships between nucleic acids and proteins form the foundational framework for molecular biology studies. In this context, obtaining high-quality nucleic acids is a crucial step for successful downstream applications. This study aimed to develop a cost-effective, eco-friendly, and versatile DNA extraction method that yields high-quality DNA from diverse biological samples. We utilized three types of borosilicate-based recycled laboratory glass as a silica source, combined with sodium iodide as a chaotropic agent, creating an efficient system for cell lysis and DNA extraction. Quality control was performed by assessing the concentration and purity of the extracted DNA using spectrophotometry, and the results were compared to those obtained with a commercial kit. DNA integrity was evaluated via agarose gel electrophoresis. To verify the suitability of the extracted DNA for downstream applications, we conducted 16S rRNA and ITS PCR amplifications. Our findings demonstrated that our DNA extraction method produced significantly higher yields, better purity, and greater integrity compared to the commercial kit. Moreover, the extracted DNA was readily applicable in PCR-based procedures, confirming the method's effectiveness for molecular biology applications.

Neurotoxin-Derived Optical Probes for Elucidating Molecular and Developmental Biology of Neurons and Synaptic Connections. Toxin-Derived Optical Probes for Neuroimaging.

Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.

Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics.

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology.

Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

A genome-scale deep learning model to predict gene expression changes of genetic perturbations from multiplex biological networks.

Systematic characterization of biological effects to genetic perturbation is essential to the application of molecular biology and biomedicine. However, the experimental exhaustion of genetic perturbations on the genome-wide scale is challenging. Here, we show TranscriptionNet, a deep learning model that integrates multiple biological networks to systematically predict transcriptional profiles to three types of genetic perturbations based on transcriptional profiles induced by genetic perturbations in the L1000 project: RNA interference, clustered regularly interspaced short palindromic repeat, and overexpression. TranscriptionNet performs better than existing approaches in predicting inducible gene expression changes for all three types of genetic perturbations. TranscriptionNet can predict transcriptional profiles for all genes in existing biological networks and increases perturbational gene expression changes for each type of genetic perturbation from a few thousand to 26945 genes. TranscriptionNet demonstrates strong generalization ability when comparing predicted and true gene expression changes on different external tasks. Overall, TranscriptionNet can systemically predict transcriptional consequences induced by perturbing genes on a genome-wide scale and thus holds promise to systemically detect gene function and enhance drug development and target discovery.

Codon optimization, expression, and purification of the reverse transcriptase enzyme variant MMLV RT2 in <i>Escherichia coli</i>

Moloney leukemia virus reverse transcriptase (MMLV RT) is an important enzyme widely utilized in molecular biology research and various diagnostic testing kits. A previous study demonstrated that variants of MMLV RT containing specific mutations contribute to enhanced stability and increased capability for RNA detection. However, the optimization and increased expression level of MMLV RT variants for large-scale production have not been extensively studied. In this research, the gene segment encoding the MMLV RT variant (E286R/E302K/L435R/D524A) was codon optimized and subcloned into pET-M expression vector. The obtained pET-M MMLV RT2 was transformed into E. coli BL21(DE3) host cell system for over-expression of the MMLV RT2 enzyme. The recombinant enzyme was then purified using nickel affinity chromatography and quantified using the Bradford method. The results revealed significant increase in recombinant enzyme yield, achieving 0.25 g/L in the culture medium. This represents a 1.4-fold increase compared to the 0.175 g/L yield reported in the previous study. Enzyme MMLV RT2 was also evaluated for its activity and was found to have comparable activity to the commercial enzyme Promega and another variant, enzyme MMLV RT1, which carries the D524A mutation and has previously been evaluated for activity. This result suggests that a high expression level of a stable MMLV-RT enzyme variant can be achieved which is beneficial for further improving the production of this important enzyme. The high yield and sustained activity of the MMLV RT2 variant indicate its potential as a reliable and cost-effective alternative for various applications in research and diagnostics. The successful optimization and expression in E. coli highlight the feasibility of large-scale production, potentially lowering costs and improving accessibility for molecular biology applications.

The Storage of the Extracted Plant DNA

A high–quality DNA has crucial importance for enzymatic applications in plant molecular biology, including agricultural research. The researchers often need to store the extracted DNA, which has to be suitable for the downstream enzymatic applications, such as a real–time PCR, even after months and years. Storing the DNA as a precipitate in ethanol at –80°C is a commonly used method of preservation. The aim of this research was to determine whether the same was applicable to a temperature of –20°C. In this study, a high-quality DNA from soybean and maize seed was extracted by the widely used CTAB method. The DNA samples were stored at three regimes: water-diluted at +4°C, water-diluted at –20°C, and an undiluted DNA in ethanol at –20°C. The concentration and yield were measured using a spectrophotometer. The impurities and fragmentation were estimated on agarose gel by electrophoresis. Additionally, the DNA was assessed amplifying by the real–time PCR. The quantity and quality of the extracted DNA were assessed prior and subsequent to an annual storage. The results indicated that ethanol provided excellent protection for a short– term DNA storage at –20°C and probably for a long–term storage due to a reduced water content and slightly basic conditions. Thus, such a storage regime could be an appropriate solution when other options are not available.

Physical Simulation-Based Calibration for Quantitative Real-Time PCR

The fluorescence quantitative polymerase chain reaction (qPCR) instrument has been widely used in molecular biology applications, where the reliability of the qPCR performance directly affects the accuracy of its detection results. In this paper, an integrated, physics-based calibration device was developed to improve the accuracy and reliability of qPCR, realizing the calibration of qPCR instruments’ standard curve through physical simulations. With this calibration device, the collected temperature was used as the control signal to alter the fluorescence output, which allowed different probes to simulate the Ct values corresponding to samples with varying initial concentrations. The temperature and optical performance of this calibration device were tested, followed by a comparative analysis comparing the on-machine test results with standard substances to assess the linearity and uniformity of the Ct values of the measured qPCR instrument. It has been proven that this physical calibration device can effectively replace the biochemical standard substance to carry out comprehensive calibration of the temperature and optical parameters of the qPCR instrument and provide a more reliable method for the periodic calibration and quality control of the qPCR instrument. This contributes to the accuracy and reliability of fluorescence qPCR instruments in the field of molecular biology.

34‐1: Invited Paper: High Throughput TFT Technology for In‐situ DNA Synthesis and Signal Sensing

By using the thin‐film transistor (TFT) technology, the oligo nucleotides array has been proved to be achieved in‐situ on a throughput matrix backplane of 16*16, with oligonucleotide lengths reaching 150mers, and the yield of each oligo has surpassed tens of fmol per pixel. This TFT‐based methods effectively balance the synthesis throughput with the loading capacity and offer a significantly lower cost for higher quality DNA for various applications in molecular biology.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

The enhancement of M13 phage titration by optimizing the origin of replication.

M13KO7, a modified M13 phage variant, carries the p15A replication origin and Tn903 kanamycin resistance gene. This study aimed to optimize M13KO7's replication by substituting the p15A origin with the higher-copy pMB1 origin (500-700 copy numbers). A 6431-nucleotide fragment from the M13KO7 plasmid lacking the p15A replication origin and kanamycin resistance gene was amplified using a long polymerase chain reaction (PCR). The modified M13AMB1 plasmid was created by adding adenine to the 3' ends of this fragment and ligating it to the pMB1-containing fragment using T/A cloning. Afterward, to prepare the phage, pM13AMB1 was transformed into E. coli TG1 bacteria, and then, using the PEG-NaCl precipitation, the modified phage was propagated. The modified phage titer was determined utilizing the serial dilution and the qPCR methods, compared with the M13KO7 phage. The results showed that in the serial dilution method, the titers of modified phage and M13KO7 phage were 4.8 × 1014 and 7 × 1012 pfu/mL, respectively. Besides, the phage titer calculated by the qPCR method for the modified phage was equal to 1.3 × 109 pfu/mL, whereas it was 4.08 × 108 pfu/mL for the M13KO7 phage. This study provides evidence that replication origin replacement led to a significant increase in phage titers. It highlights the importance of replication optimization for molecular biology applications.

Comparative Analysis of 1,9-Dimethyl-Methylene Blue and Toluidine Blue Interactions with ct-DNA, G-Quadruplex DNA, and ssDNA

This study presents a comprehensive investigation into the interactions of two distinct synthetic dyes—1,9-Dimethyl-Methylene Blue and Toluidine Blue —with different DNA structures, namely calf thymus DNA (ct-DNA), G-quadruplex DNA, and single stranded DNA (ssDNA). The objective of this research is to elucidate the molecular affinities of these dyes for specific DNA structures and explore their potential applications in molecular biology and diagnostics. The experimental approach involved detailed UV-visible spectroscopic analyses, to probe the binding affinities of each dye with ct-DNA, G-quadruplex DNA, and ssDNA. The study aimed to assess the selectivity of these dyes towards the unique structural features of each DNA entity. The binding stoichiometry is defined from Job’s method. The selectivity of the dyes towards DNA also investigated with competitive dialysis experiments. The binding stoichiometry were 1:1 for 1,9-Dimethyl-Methylene Blue and Toluidine Blue and G-quadruplex DNA or ssDNA. Besides, results indicate that 1,9-Dimethyl-Methylene Blue and Toluidine Blue exhibit a pronounced affinity for G-quadruplex DNA, and ct-DNA. While single-stranded DNA is a fundamental component of DNA replication and transcription, our dyes exhibit lower affinity for this structure. The selectivity is advantageous, as it allows for the discrimination between single-stranded and structured DNA regions. The potential applications in studying DNA dynamics and unwinding processes are vast.