Microscopic DNA Sequence Visualization in a Microfluidic Device

Kyubong JO 한국생물공학회 2023 한국생물공학회 학술대회 Vol.2023 No.10

Elongation and migration of single DNA molecules in microchannels using oscillatory shear flows

Jo, Kyubong,Chen, Yeng-Long,de Pablo, Juan J.,Schwartz, David C. Royal Society of Chemistry 2009 Lab on a chip Vol.9 No.16

<P>Much of modern biology relies on the strategic manipulation of molecules for creating ordered arrays prior to high throughput molecular analysis. Normally, DNA arrays involve deposition on surfaces, or confinement in nanochannels; however, we show that microfluidic devices can present stretched molecules within a controlled flow in ways complementing surface modalities, or extreme confinement conditions. Here we utilize pressure-driven oscillatory shear flows generated in microchannels as a new way of stretching DNA molecules for imaging “arrays” of individual DNA molecules. Fluid shear effects both stretch DNA molecules and cause them to migrate away from the walls becoming focused in the centerline of a channel. We show experimental findings confirming simulations using Brownian dynamics accounting for hydrodynamic interactions between molecules and channel-flow boundary conditions. Our findings characterize DNA elongation and migration phenomena as a function of molecular size, shear rate, oscillatory frequency with comparisons to computer simulation studies.</P> <P>Graphic Abstract</P><P>This paper reports the development of a novel and simple way for presenting stretched DNA molecules in microchannels driven by oscillatory shear flow. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=b902292a'> </P>

Microscopic DNA Sequence Analysis without Sequencing

Kyubong JO 한국생물공학회 2019 한국생물공학회 학술대회 Vol.2019 No.10

A single-molecule barcoding system using nanoslits for DNA analysis : nanocoding.

Jo, Kyubong,Schramm, Timothy M,Schwartz, David C Humana Press 2009 METHODS IN MOLECULAR BIOLOGY -CLIFTON THEN TOTOWA- Vol.544 No.-

<P>Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.</P>

mRNA 백신의 화학

조규봉 ( Kyubong Jo ) 한국현장과학교육학회 2021 현장과학교육 Vol.15 No.3

코로나19 팬데믹에서 화이자, 모더나사에서 개발하여 판매하고 있는 mRNA 백신은 현재 가장 신뢰받는 백신이지만 mRNA 백신은 2020년에 처음 허가되어 이용되고 있는 새로운 의약품이다. 세포 밖에서 mRNA를 넣는 기술은 1991년 시작되어 이를 의약품으로 개발하는 데 30년이 필요하였으며 여러 사람들의 노력에 의해서 지금의 의약품으로 만들어질 수 있었다. 주목할 만한 두가지 기술은 선천면역 반응을 피하기 위한 변형된 핵산을 찾아내는 과정과 지질 나노입자를 이용한 약물전달 기술로써 현재의 mRNA 백신 기술을 완성하는데에 핵심적인 역할을 하였다. 또한 mRNA 약물전달 기술은 백신뿐 아니라 암을 치료하고 노화된 세포를 젊게 만들 수 있는 새로운 기술로써 머지 않은 미래에 의학기술의 혁명을 가져올 것으로 기대되고 있다. 이 글에서는 mRNA 백신에 대한 화학 기술에 대해서 살펴보고자 한다.

Neutravidin coated surfaces for single DNA molecule analysis

Kim, Yoori,Jo, Kyubong Royal Society of Chemistry 2011 Chemical communications Vol.47 No.22

<P>We present a novel approach for single DNA molecule analysis using neutravidin coated surfaces. DNA molecules are elongated and reversibly immobilized on neutravidin coated surfaces with pH and salt controls. We demonstrate restriction enzyme reactions for optical mapping and ligation for tethered DNA molecules.</P> <P>Graphic Abstract</P><P>Microfluidic flow dynamically elongates 110 μm DNA biotin-tethered on neutravidin coated surfaces. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c0cc05396a'> </P>

Microfluidic Device to Maximize Capillary Force Driven Flows for Quantitative Single-Molecule DNA Analysis

Kim Taesoo,Jo Kyubong 한국바이오칩학회 2023 BioChip Journal Vol.17 No.3

Microfl uidics is fl ourishing due to its signifi cant applications in life sciences and biomedical engineering. One of the key challenges in microfl uidics is the manipulation and control of fl uids within microscale channels. Capillary force-driven fl ows provide a potential solution to this challenge by eliminating the need for an external power source. Capillary force- driven fl ows are particularly useful for the reproducible and reliable quantitative analysis of single-molecule DNA. We have designed several microfl uidic devices that employ capillary force to enhance the deposition of DNA molecules onto a positively charged glass surface from a sample solution. The optimization of specifi c dimensions within the microfl uidic device resulted in increased effi ciency of DNA deposition. Shortening the microchannel length reduced fl ow resistance and decreasing the microchannel height enhanced capillary force. Additionally, increasing the outlet reservoir capacity achieves mechanical equilibrium in situations where fl uid fl ow is maximized. These optimizations served to maximize capillary force and improve DNA deposition on the glass surface. The developed device represents an ultra-sensitive platform for quantitative DNA analysis and rapid, accurate point-of-care testing with a minimum detection limit achieved. In conclusion, our work demonstrates the potential of capillary force-driven microfl uidics for the reproducible and effi cient manipulation of fl uids within microscale channels.

Tamra-Polypyrrole for A/T Sequence Visualization on DNA Molecules

Lee, Seonghyun,Jo, Kyubong Published for the Biophysical Society by the Rocke 2019 Biophysical journal Vol.116 No.3

Molecular Propulsion: Chemical Sensing and Chemotaxis of DNA Driven by RNA Polymerase

Yu, Hua,Jo, Kyubong,Kounovsky, Kristy L.,Pablo, Juan J. de,Schwartz, David C. American Chemical Society 2009 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.131 No.16

<P>Living cells sense extracellular signals and direct their movements in response to stimuli in environment. Such autonomous movement allows these machines to sample chemical change over a distance, leading to chemotaxis. Synthetic catalytic rods have been reported to chemotax toward hydrogen peroxide fuel. Nevertheless individualized autonomous control of movement of a population of biomolecules under physiological conditions has not been demonstrated. Here we show the first experimental evidence that a molecular complex consisting of a DNA template and associating RNA polymerases (RNAPs) displays chemokinetic motion driven by transcription substrates nucleoside triphosphates (NTPs). Furthermore this molecular complex exhibits a biased migration into a concentration gradient of NTPs, resembling chemotaxis. We describe this behavior as 'Molecular Propulsion', in which RNAP transcriptional actions deform DNA template conformation engendering measurable enhancement of motility. Our results provide new opportunities for designing and directing nanomachines by imposing external triggers within an experimental system.</P>

Zinc-finger motif noncovalent interactions with double-stranded DNA characterized by negative-ion electrospray ionization mass spectrometry

Park, Soojin,Jo, Kyubong,Oh, Han Bin Royal Society of Chemistry 2011 The Analyst Vol.136 No.18

<P>A zinc-finger motif recognizes specific sequences on the double helical structure of DNA. This sequence recognition property offers great promise for various biotechnology applications. Accordingly, it is crucially important to characterize zinc-finger binding characteristics for further developments. Although the gel shift assay or phage display is traditionally used for determining the binding characteristics of zinc-fingers for double stranded DNA, in the present study we utilize electrospray ionization mass spectrometry as an advanced and convenient characterization tool because of the rich information it provides, and its quantitative sensitivity, operational simplicity, and no need for radioactive labeling. Here we demonstrate the use of negative-ion electrospray ionization mass spectrometry for competition-based quantitative comparison of the zinc-finger motif sequence specificity, stoichiometry, and metal ion dependence.</P> <P>Graphic Abstract</P><P>The DNA recognition by zinc-fingers is systematically studied using negative-ion electrospray ionization mass spectrometry. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c1an15376e'> </P>