aOceanography Section,Science Research Center,Kochi University,Kochi,Japan

bThe Graduate University for Advanced Studies,Kanagawa,Japan

cDepartment of Mechanical Engineering,The University of Tokyo,Tokyo,Japan

Single molecular DNA/RNA sequencing with microscopy

Masanori Kataokaa,*,Kuniaki Nagayamab,Hidehiro Oanac

aOceanography Section,Science Research Center,Kochi University,Kochi,Japan

bThe Graduate University for Advanced Studies,Kanagawa,Japan

cDepartment of Mechanical Engineering,The University of Tokyo,Tokyo,Japan

A R T I C L E I N F O

Article history:

Available online 25 November 2015

Single molecular DNA/RNA

sequencing

Quantum dot complex

Ultra high-throughput DNA sequencing has been a very hot topic beyond the feld of genomic researches.There have been various approaches to this issue ranging from direct observation of individual DNA synthesis[1],amperometric/optical detection of bases using a nanopore[2,3]and direct sequencing with a high-resolution probe microscope[4].Recently,we have developed a technique to chemically modify all nucleobases in DNA[5],so that the bases A,T,G,C can be differentiated in the sequence by using high-resolution electron microscopy(EM).The requirements toward this sequencing, which relies on the EM capability are;(i)stretching of singlestranded DNA,(ii)high yield structural modifcation without damage in DNA and(iii)a way to protect or reduce the electron dose damage to bases.The most crucial step particularly for the case of observing intact organic molecules without relying on labeled heavy elements is the last requirement and we are still underway to fnd how to do it.In this paper we will report what we have done for the frst two requirements.There are two ways to have stretched and modifed single-stranded DNA molecules;stretch“before”modifcation or“after,”but we found that DNA strands become fragile and break easily,so that“stretch before”scheme is employed in this paper.

DNA(48.5 kb,16.5 mm)was employed as the sample.DNA was frst stretched out and immobilized onto anamorphous carbon thin layer or anamorphous carbon flm(micro grid)by molecular combing.Then the fxed DNA strands were exposed to 1.75 M chloroacetaldehyde in the acetate buffer for modifcation of adenosine.To visualize the degree of etheno adduct, anti-ethenoadenosine antibody was added,which was fuoresceinated by a secondary antibody carrying Qdot.Then, the solid surface was rinsed with PBS buffer and observation was carried out using AFM and EM.The result of AFM imaging for the stretched and modifed DNA is shown in Fig.1A.From the size analysis,the dots in the photo are identifed as Qdots, which cover the entire length of DNA that extends about 20 mm. As seen in the uppermost(most enlarged)photo,DNA is labeled by Qdots rather uniformly,with the spacing of several 10 nm. Considering that the size of an antibodies-Qdotcomplex is about 20 nm in diameter,they are packed densely along the DNA. The observation of TEM carried out for the same sample is shown in Fig.1B.The high-resolution TEM picture shows that the Qdotcomplex is lined on a DNA backbone and a detailed shape of Qdots.These results demonstrate a possibility of a TEM sequencer.

Fig.1–Example AFM image of adenine-labeled DNA molecules with ethenoadenosine antibodies and Qdot-conjugated secondary antibodies(tapping mode)(A)and TEM picture of adenine-labeled DNA molecules with ethenoadenosine antibodies and Qdot-conjugated secondary antibodies(STEM/HAADF image)(B).

R E F E R E N C E S

[1]Eid J,Fehr A,Gray J,et al.Real-time DNA sequencing from single polymerase molecules.Science 2009;323: 133–138.

[2]Singer A,Wanunu M,Morrison W,et al.Nanopore based sequence specifc detection of duplex DNA for genomic profling.Nano Lett 2010;10:738–742.

[3]McNally B,Singer A,Yu Z,et al.Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays.Nano Lett 2010;10:2237–2244.

[4]Tanaka H,Kawai T.Partial sequencing of a single DNA molecule with a scanning tunnelling microscope.Nat Nanotechnol 2009;4:518–522.

[5]Kataoka M,Nagayama K.Method for modifcation of nucleotides in nucleic acid,and nucleic acid having modifed nucleotide therein.WO/2009/020249,2009.

*E-mail address:m.kataoka@kochi-u.ac.jp.

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2015.11.113

1818-0876/?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).