IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-10 DOI: 10.1016/j.ymeth.2025.09.004

Jarrod Laro , Monica Ness , Joseane Godinho , Randy Coats , Laura-Isobel McCall

{"title":"Optimization of 13C stable isotope labeling for the study of tricarboxylic cycle intermediates in mouse models","authors":"Jarrod Laro , Monica Ness , Joseane Godinho , Randy Coats , Laura-Isobel McCall","doi":"10.1016/j.ymeth.2025.09.004","DOIUrl":"10.1016/j.ymeth.2025.09.004","url":null,"abstract":"<div><div>The tricarboxylic acid cycle (TCA), also known as the Krebs Cycle or the citric acid cycle, is an essential metabolic pathway involved in energy production that is often impacted by disease, making it of key interest to identify effective, affordable, and simple ways to monitor the impact of disease on TCA metabolism. <sup>13</sup>C-based stable isotope labeling is a useful technique to track pathway alterations in living hosts. However, infusion-based methodologies are slow and expensive despite achieving steady-state labeling. Bolus-based methods are cheaper, faster, and compatible with biohazardous models, but require optimization to achieve maximum labeling. Herein, we performed bolus-based stable isotope labeling experiments in mouse models to identify the optimal dosage amount, label administration length, fast length prior to label administration, <sup>13</sup>C-labeled precursor, and route of administration for the TCA cycle in the esophagus, heart, kidney, liver, plasma, and proximal colon. <sup>13</sup>C-glucose at a concentration of 4 mg/g administered via intraperitoneal injection followed by a 90 min label incorporation period achieved the best overall TCA labeling. For most organs, a 3 h fast prior to label administration improved labeling, but labeling in the heart was better with no fasting period, showcasing the need to optimize methodology on an organ-by-organ basis. We also identified that bolus administration of glucose provided little impact on metabolism compared to vehicle control. The experiments outlined here provide critical information for designing in vivo stable isotope labeling experiments for the study of the TCA cycle.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 75-81"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145051513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Advancing antisense oligonucleotide delivery through click chemistry based chemical conjugation with designed short non-cationic peptides for Duchenne muscular dystrophy 通过点击化学与设计的短非阳离子肽的化学偶联，推进反义寡核苷酸递送治疗杜氏肌营养不良。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-15 DOI: 10.1016/j.ymeth.2025.09.005

Surojit Ghosh , Mohammad Umar Arshi , Satyajit Ghosh , Moumita Jash , Nabanita Mukherjee , Aniket Jana , Arun Shastry , Deepika Karanth , Khushbu Nishad , Nirmal Kumar Rana , Sudipta Bhattacharyya , Surajit Ghosh

{"title":"Advancing antisense oligonucleotide delivery through click chemistry based chemical conjugation with designed short non-cationic peptides for Duchenne muscular dystrophy","authors":"Surojit Ghosh , Mohammad Umar Arshi , Satyajit Ghosh , Moumita Jash , Nabanita Mukherjee , Aniket Jana , Arun Shastry , Deepika Karanth , Khushbu Nishad , Nirmal Kumar Rana , Sudipta Bhattacharyya , Surajit Ghosh","doi":"10.1016/j.ymeth.2025.09.005","DOIUrl":"10.1016/j.ymeth.2025.09.005","url":null,"abstract":"<div><div>Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disease caused by frame shift mutations in the gene encoding dystrophin. 2́-O-methyl phosphorothioate (2′-OMePS) serves as an antisense RNA platform clinically used in DMD patients to facilitate exon skipping and production of an internally truncated, yet functional dystrophin protein. Effective delivery and uptake of antisense oligonucleotides (ASOs) by target cells are crucial for their efficacy. Peptide-conjugated ASOs offer a promising next-generation platform, where a cell-penetrating peptide (CPP) is linked to the 2′-OMePS backbone to enhance cellular uptake. Herein, we designed and synthesized a new non-cationic short CPP sequence that can be efficiently conjugated with the negatively charged 2′-OMePS ASO backbone using click chemistry. Conjugation of the lead peptide ETWWK to 2′-OMePS ASO resulted in significant cellular internalization with precise nuclear localization of the ASO cargo. Cellular uptake was assessed in C2C12 and human DMD patient-derived myoblast cells via fluorescence microscopy and flow cytometry. Additionally, the synthesized ETWWK-ASO conjugate exhibits a significant 1.94 fold upregulation of dystrophin protein in the clinically relevant DMD patient-derived cell line. Our findings suggest that the identified peptide holds promise for facilitating ASO delivery at the site of splicing. This study highlights the efficient conjugation of CPPs to negatively charged 2′-OMePS ASO through tailored conjugation strategies, and will eventually be a therapeutic avenue for future ASO-based DMD treatments.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 92-107"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145079293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analytical capability of Raman spectroscopy to detect biochemical changes in red blood cell membrane disorders 拉曼光谱检测红细胞膜紊乱生化变化的分析能力。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-20 DOI: 10.1016/j.ymeth.2025.09.007

Panchanil Sarmah , Ruchee Khanna , Aseefhali Bankapur

{"title":"Analytical capability of Raman spectroscopy to detect biochemical changes in red blood cell membrane disorders","authors":"Panchanil Sarmah , Ruchee Khanna , Aseefhali Bankapur","doi":"10.1016/j.ymeth.2025.09.007","DOIUrl":"10.1016/j.ymeth.2025.09.007","url":null,"abstract":"<div><div>Detection of RBC membrane disorders with currently available modalities, such as osmotic fragility test (OFT), EMA binding test, ektacytometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and next-generation sequencing (NGS), has been challenging as they either lack biochemical inferences or are complex in nature. Raman spectroscopy, a highly analytical method known to produce molecular fingerprints, has proven potential in extracting biochemical information from single individual cells. Recent advancements in membrane-targeted Raman measurements using excitation spots of donut and line intensity profiles can transform lab-on-chip Raman-activated cell sorting methods into a potential technique for RBC membrane disorder diagnosis. We, therefore, conjecture that Raman spectroscopy can be a strong contender as a diagnostic modality in RBC membranopathies. In this comprehensive review<strong>,</strong> we have attempted to encompass the disorder-specific molecular defects, present diagnostic modalities, and their limitations, and explored the translational possibility of Raman spectroscopy as a diagnostic tool for membranopathies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 118-133"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

ResDeepGS: A deep learning-based method for crop phenotype prediction ResDeepGS：基于深度学习的作物表型预测方法。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-08 DOI: 10.1016/j.ymeth.2025.07.013

Chaokun Yan , Jiabao Li , Qi Feng , Junwei Luo , Huimin Luo

{"title":"ResDeepGS: A deep learning-based method for crop phenotype prediction","authors":"Chaokun Yan , Jiabao Li , Qi Feng , Junwei Luo , Huimin Luo","doi":"10.1016/j.ymeth.2025.07.013","DOIUrl":"10.1016/j.ymeth.2025.07.013","url":null,"abstract":"<div><div>Genomic selection (GS) is a breeding technique that utilizes genomic markers to predict the genetic potential of crops and animals. This approach holds significant promise for accelerating the improvement of agronomic traits and addressing food security challenges. While traditional breeding methods based on statistical or machine learning techniques have been useful in predicting traits for some crops, they often fail to capture the complex interactions between genotypes and phenotypes. Additionally, these methods struggle to handle large-scale data, limiting their predictive performance. Recent advancements in deep learning offer a promising solution by better capturing nonlinear relationships and gene interactions.</div><div>In this study, we propose a novel crop phenotype prediction method, ResDeepGS, which leverages deep learning techniques. The model consists of two main components: the feature selection module and the phenotype prediction module. The feature selection module employs an incremental recursive feature elimination method, combining the strengths of recursive feature elimination and incremental learning to improve both the efficiency and reliability of feature selection. The phenotype prediction module integrates an enhanced multi-layer convolutional neural network with residual structures and dropout strategies to better capture complex relationships in gene data, accelerate convergence, and reduce overfitting. Through extensive experimentation, we demonstrate that ResDeepGS outperforms current state-of-the-art methods on three datasets: wheat, maize, and soybean. Notably, on the wheat dataset, ResDeepGS improved prediction accuracy by 5% to 9%, highlighting its superior performance in genomic selection tasks. These results underscore the robustness and adaptability of ResDeepGS, offering a promising solution for enhancing crop breeding efficiency and addressing future food security challenges.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 65-74"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Efficient RNA nucleotide encoding enhances the accurate prediction of ac4C modifications 高效的RNA核苷酸编码增强了对ac4C修饰的准确预测。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-08-31 DOI: 10.1016/j.ymeth.2025.07.012

Na Li , Xiao Wang , Ming Zeng , Feng Cao , Ke Qiu , Jianbo Qiao

引用次数: 0

Gelatin methacryloyl − thiolated graphene oxide composites for soft tissue engineering 明胶甲基丙烯酰硫化氧化石墨烯复合材料用于软组织工程。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-10-11 DOI: 10.1016/j.ymeth.2025.10.003

Ayoub Shiravandi , Ibrahim Zarkesh , Melina Ghasemian , Mohammad Kazemi Ashtiani , Gholamreza Bayazian , Alimohamad Asghari , Rafieh Alizadeh , Zohreh Bagher , Seyed Mohammad Davachi

{"title":"Gelatin methacryloyl − thiolated graphene oxide composites for soft tissue engineering","authors":"Ayoub Shiravandi , Ibrahim Zarkesh , Melina Ghasemian , Mohammad Kazemi Ashtiani , Gholamreza Bayazian , Alimohamad Asghari , Rafieh Alizadeh , Zohreh Bagher , Seyed Mohammad Davachi","doi":"10.1016/j.ymeth.2025.10.003","DOIUrl":"10.1016/j.ymeth.2025.10.003","url":null,"abstract":"<div><div>Soft tissue engineering represents a transformative solution for regenerating damaged tissues, with the development of advanced hydrogels playing a crucial role in this process. Gelatin methacryloyl (GelMA) has emerged as a promising material due to its biocompatibility and ability to support cell growth; however, its mechanical limitations have driven the exploration of composite hydrogels, with thiolated graphene oxide (tGONS) standing out as a key additive. In this study, we present a novel strategy involving thiolated graphene oxide nanosheets (tGONS) as covalent crosslinkers within the GelMA hydrogel matrix, wherein thiol groups on tGONS react with GelMA’s acrylate groups, enhancing crosslinking and strengthening the hydrogel network. The results demonstrate that incorporation of tGONS at optimized concentrations (≤0.5 mg/mL) leads to a substantial increase in Young’s modulus and gel fraction, along with a marked enhancement in antioxidant activity, evidenced by up to 40 % DPPH and 60 % H<sub>2</sub>O<sub>2</sub> scavenging in G1GTII formulations. Despite the increased stiffness, acceptable elongation and high cytocompatibility (>85 % cell viability) were maintained. Additionally, the composite hydrogels showed improved resistance to enzymatic degradation and reduced swelling, offering more precise control over physicochemical properties.These findings highlight the multifunctional advantages of GelMA–tGONS composite hydrogels, positioning them as adaptable and robust scaffolds suitable for applications in soft tissue engineering, including cartilage, dermal, and neural regeneration.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 170-181"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145285367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Novel primer-less amplification method for detection of RNA molecules 新型无引物扩增方法检测RNA分子。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-10-16 DOI: 10.1016/j.ymeth.2025.10.007

Alicia del Prado , María I. Martínez-Jiménez , Luis Blanco, Miguel de Vega

{"title":"Novel primer-less amplification method for detection of RNA molecules","authors":"Alicia del Prado , María I. Martínez-Jiménez , Luis Blanco, Miguel de Vega","doi":"10.1016/j.ymeth.2025.10.007","DOIUrl":"10.1016/j.ymeth.2025.10.007","url":null,"abstract":"<div><div>As shown here, isothermal and primer-less amplification of specific padlock probes allows direct detection of SARS-CoV2 RNA without needing for a reverse transcription step. This simplified method of Hyperbranched Rolling Circle Amplification (HRCA) only requires three enzymes: SplintR (to ligate a specific padlock probe to its circular form, only when viral RNA is present), the unique DNA primase <em>Tth</em>PrimPol (to generate <em>de novo</em> DNA primers), and an engineered and thermostabilized variant of bacteriophage phi29 DNA polymerase (phi29 DNApol), named Qx5. Qx5 is significantly more efficient than wild-type phi29 DNApol and shows an unexpectedly strong 3′-5′ exoribonucleolytic activity capable of trimming the 3′ polyA tail of the RNA target, thus enabling it as a primer for Qx5 to start Rolling Circle Amplification (RCA) of a nearby circularized padlock. The RCA step, that yields a long ssDNA concatemeric product (RCA product, RCP) is coupled to a second isothermal amplification step, assisted by <em>Tth</em>PrimPol by synthesizing DNA primers on the RCP, that triggers exponential HRCA of the padlock sequence, catalyzed by Qx5. As a proof of concept, the application of this method for detection of SARS-CoV2 RNA rendered a significant amount of DNA in just 2 h at 37 °C, that can be easily evidenced by colorimetry, or even quantitated with a pocket fluorometer, and served for a quick diagnosis of SARS-CoV2 RNA infected <em>versus</em> non-infected samples.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 227-239"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Michaelis-Menten kinetics of RasGAP proteins by a rapid fluorescence-based assay RasGAP蛋白的Michaelis-Menten动力学

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-08 DOI: 10.1016/j.ymeth.2025.09.003

Maxum E. Paul , Kimberly J. Vish , Titus J. Boggon

{"title":"Michaelis-Menten kinetics of RasGAP proteins by a rapid fluorescence-based assay","authors":"Maxum E. Paul , Kimberly J. Vish , Titus J. Boggon","doi":"10.1016/j.ymeth.2025.09.003","DOIUrl":"10.1016/j.ymeth.2025.09.003","url":null,"abstract":"<div><div>Ras small GTPases are essential for a wide range of cellular processes. These proteins cycle between the GDP-loaded and GTP-loaded states, and the actions of GTPase activating proteins (GAPs) are necessary to stimulate Ras-mediated GTP hydrolysis. Here, we provide a protocol to achieve Michaelis-Menten kinetic profiling of GAP-mediated stimulation of a small GTPase by real-time monitoring of inorganic phosphate release <em>in vitro</em>. This is achieved using fluorescence of the Phosphate Sensor protein, an MDCC conjugate with periplasmic phosphate binding protein (PstS). We use H-Ras small GTPase pre-loaded with GTP and its stimulation by p120RasGAP (RasGAP, RASA1) as an example of this protocol. We discuss protocol design, assay development, data collection, processing, and analysis. Typical assays comprise up to twenty simultaneous reactions with phosphate production rates on the order of tens of nM/s. We also provide guidelines for the optimization of reagent conditions, particularly salt concentrations, and assess their functional impact. The described protocol provides a convenient and comprehensive method to achieve accurate monitoring of small GTPase activation by GAP proteins using widely available materials and suitable to a range of applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 108-117"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Unveiling the pathogenicity of allosteric protein mutations via multifaceted feature ensembling 通过多面特征集合揭示变构蛋白突变的致病性。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-08 DOI: 10.1016/j.ymeth.2025.07.014

Huiling Zhang , Xijian Li , Junwen Huang , Yuetong Li , Shaozhen Cai , Haiyan Wang , Yanjie Wei

{"title":"Unveiling the pathogenicity of allosteric protein mutations via multifaceted feature ensembling","authors":"Huiling Zhang , Xijian Li , Junwen Huang , Yuetong Li , Shaozhen Cai , Haiyan Wang , Yanjie Wei","doi":"10.1016/j.ymeth.2025.07.014","DOIUrl":"10.1016/j.ymeth.2025.07.014","url":null,"abstract":"<div><div>Allostery proteins play a central role in biological processes and systems. Uncovering the biological effects of allosteric protein mutations and their role in disease progression remains a significant challenge. Theoretically, computational approaches hold the potential to enable large-scale interpretation of genetic variants in allosteric proteins. Nevertheless, general-purpose variant effect prediction (VEP) methodologies overlook the characteristic disparities across different genes. What is more critical is that individual tools frequently display inconsistencies, biases, and fluctuations in quality. Consequently, the predictions obtained from existing VEP approaches are considered insufficiently reliable. In the present research, we constructed an a multifaceted-feature-based ensemble learning approach to forecast the pathogenicity of missense mutations within allosteric proteins. The proposed method used categorical boosting to integrate four types of features, namely, sequence information, AlphaFold2-extracted biochemical properties, prediction scores from other VEP methods, and allele frequency from gnomAD. Our method demonstrated superior performance with an AUC of 0.912 when tested on a benchmark allosteric protein dataset, outperforming 22 general VEP methods. To facilitate the identification of pathogenic mutations in the sea of rare variants discovered as sequencing studies expand on a large scale, we provided the pathogenicity probabilities of all potential amino acid substitutions in 202 allosteric-protein-encoding genes. To sum up, our research indicates that multifaceted-feature-based ensemble learning models can offer valuable independent evidence for interpreting missense mutations in allosteric proteins, which will be broadly applicable in both research and clinical contexts.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 82-91"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strain promoted click labeling of oligonucleotides on solid-phase support 应变促进在固相载体上的寡核苷酸点击标记。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-06 DOI: 10.1016/j.ymeth.2025.09.001

Michaela Beranova , Petra Grossova , Jiri Demuth , Filip Kostelansky , Veronika Novakova , Petr Zimcik , Miroslav Miletin

{"title":"Strain promoted click labeling of oligonucleotides on solid-phase support","authors":"Michaela Beranova , Petra Grossova , Jiri Demuth , Filip Kostelansky , Veronika Novakova , Petr Zimcik , Miroslav Miletin","doi":"10.1016/j.ymeth.2025.09.001","DOIUrl":"10.1016/j.ymeth.2025.09.001","url":null,"abstract":"<div><div>Chemically modified oligonucleotides (ONs) are essential tools in molecular biology, diagnostics, and therapeutics. Strain-promoted azide–alkyne cycloaddition (SPAAC) offers an efficient and bioorthogonal method for ON functionalization. While SPAAC reactions on solid-phase support provide distinct advantages, particularly for the incorporation of lipophilic labels, factors influencing their efficiency remain poorly characterized. The interplay between the physicochemical properties of the modifying molecule, the nature of the solid support, and the labeling site within the ON chain has not been systematically evaluated. In this study, we systematically investigate how modifying molecule properties (size, polarity) and concentration, solid support type, labeling site within the ON chain, and reaction time influence efficiency of labeling. Our findings demonstrate that while polar modifying molecules react efficiently across all solid supports, lipophilic molecules can exhibit reduced reactivity on glass-based supports, particularly in positions close to the 3́-end of the oligonucleotide attached to the support. We further show that conjugation at the 5′-terminus consistently yields the highest efficiencies, with a gradual decline observed as the modification site approaches the 3′-end. A 1 mM concentration of labeling reagent was sufficient to achieve high yields on polystyrene support for all labels and on CPG 500 for the polar labels. The size of the modifying molecule had a lesser effect compared to other factors. The method also benefits from recovery and reusability of the unreacted label. These results enable the rational design of efficient ON labeling protocols on solid-phase support while minimizing reagent consumption, contributing to both cost-effectiveness and environmental sustainability.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 46-54"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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