{"title":"Multiplexed stamp-transfer AFM deposition improves resolution of protein-DNA conformational states.","authors":"Emily Lentz,Zimeng Li,Corey Davis,Dorothy Erie","doi":"10.1016/j.bpj.2025.06.027","DOIUrl":null,"url":null,"abstract":"Single-molecule analysis of atomic force microscopy (AFM) images is a powerful tool for characterizing the structural and conformational properties of proteins, DNA, and protein-DNA complexes, as well as nonbiological molecules, such as polymers. Since the invention of AFM in 1986, significant technical advances have been made, including faster scan speeds and automated image collection and analysis. Deposition methods, however, remain essentially unchanged. Typically, several microliters of the sample are dropped onto a mica surface (unmodified or modified), allowed to spread, rinsed with water, and dried. Although this method is generally effective, it remains a chokepoint to efficiently collecting AFM data. To alleviate this bottleneck, we invented a stamp-transfer method to deposit multiple samples simultaneously onto a mica surface for imaging. We fabricate arrays of microwells in a silicon chip, fill them with samples, and bring the silicon chip into soft contact with mica to transfer the sample. This method not only allows the simultaneous deposition of multiple different protein and DNA samples, but it also expands the buffer conditions for deposition of DNA and protein-DNA complexes onto an unmodified the mica surface into the physiological-salt range. Furthermore, our data indicate that the stamp-transfer deposition significantly improves the ability to resolve different conformational states of protein-DNA complexes from one another. Finally, this method can be readily automated and has the potential revolutionize AFM imaging both by improving resolution and making it \"high throughput\".","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":"26 1","pages":""},"PeriodicalIF":3.2000,"publicationDate":"2025-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biophysical journal","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/j.bpj.2025.06.027","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
Single-molecule analysis of atomic force microscopy (AFM) images is a powerful tool for characterizing the structural and conformational properties of proteins, DNA, and protein-DNA complexes, as well as nonbiological molecules, such as polymers. Since the invention of AFM in 1986, significant technical advances have been made, including faster scan speeds and automated image collection and analysis. Deposition methods, however, remain essentially unchanged. Typically, several microliters of the sample are dropped onto a mica surface (unmodified or modified), allowed to spread, rinsed with water, and dried. Although this method is generally effective, it remains a chokepoint to efficiently collecting AFM data. To alleviate this bottleneck, we invented a stamp-transfer method to deposit multiple samples simultaneously onto a mica surface for imaging. We fabricate arrays of microwells in a silicon chip, fill them with samples, and bring the silicon chip into soft contact with mica to transfer the sample. This method not only allows the simultaneous deposition of multiple different protein and DNA samples, but it also expands the buffer conditions for deposition of DNA and protein-DNA complexes onto an unmodified the mica surface into the physiological-salt range. Furthermore, our data indicate that the stamp-transfer deposition significantly improves the ability to resolve different conformational states of protein-DNA complexes from one another. Finally, this method can be readily automated and has the potential revolutionize AFM imaging both by improving resolution and making it "high throughput".
期刊介绍:
BJ publishes original articles, letters, and perspectives on important problems in modern biophysics. The papers should be written so as to be of interest to a broad community of biophysicists. BJ welcomes experimental studies that employ quantitative physical approaches for the study of biological systems, including or spanning scales from molecule to whole organism. Experimental studies of a purely descriptive or phenomenological nature, with no theoretical or mechanistic underpinning, are not appropriate for publication in BJ. Theoretical studies should offer new insights into the understanding ofexperimental results or suggest new experimentally testable hypotheses. Articles reporting significant methodological or technological advances, which have potential to open new areas of biophysical investigation, are also suitable for publication in BJ. Papers describing improvements in accuracy or speed of existing methods or extra detail within methods described previously are not suitable for BJ.