Tag Archives: AFM Tips

DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices

MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA–protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins.*

In the article “DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices” Ming Liu, Saeid Movahed, Saroj Dangi, Hai Pan, Parminder Kaur, Stephanie M. Bilinovich, Edgar M. Faison, Gage O. Leighton, Hong Wang, David C. Williams Jr. and Robert Riehn demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), they determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops.*

Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernible large-scale impact of protein binding. The authors of the article demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.*

For atomic force microscopy (AFM), the authors used a 7,163-bp linear DNA substrate which contains a 1,697-bp methylated CpG-rich region that is flanked by 2,742-bp and 2,724-bp CpG-free regions. For MeCP2, the DNA substrate and the protein were diluted in AFM imaging buffer (HEPES 20 mM, Mg(OAc)210mM, NaCl 100mM, pH 7.5), mixed together and deposited on freshly peeled mica. For MBD2FLsc, the authors describe how they first mixed the protein and DNA and then diluted the sample in AFM buffer before deposition. The final MeCP2 concentration deposited on mica was 7.5nM, and the MBD2FLsc concentration was 14nM. The mica samples were then washed with filtered deionized water and dried with nitrogen.*

NANOSENSORS™ PointProbe® Plus PPP-FMR AFM probes ( ≈2.8N/m) were used to image the sample at a scan resolution of 5.9nm and a scan rate of 3μm/s.*

Figure 6 from “DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices “ by Ming Liu et al.:
Atomic force microscopy (AFM) of methylated substrates under various conditions. AFM of bare methylated dsDNA oligomer a, the same oligomer with MBD2Flsc b, and with MeCP2 c. Scale bars are 200nm. The green arrows point at ends, and cyan arrows point at loops. The inset in d illustrates the counting method for loops. The distribution of number of free ends (d) and the distribution of number of loops (e) for DNA or DNA–protein complexes was determined from such images (bare DNA N=118, MBD2FLsc N=98, MeCP2 N=108). f Height of isolated DNA–DNA crossings (bare DNA N=52, MBD2FLsc N=68, MeCP2 N=83)

*Ming Liu, Saeid Movahed, Saroj Dangi, Hai Pan, Parminder Kaur, Stephanie M. Bilinovich, Edgar M. Faison, Gage O. Leighton, Hong Wang, David C. Williams Jr. and Robert Riehn
DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices
Epigenetics & Chromatin volume 13, Article number: 18 (2020)
DOI: https://doi.org/10.1186/s13072-020-00339-7

Please follow this external link to read the full article: https://rdcu.be/b3iTm

Open Access: The article “DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices” by Ming Liu, Saeid Movahed, Saroj Dangi, Hai Pan, Parminder Kaur, Stephanie M. Bilinovich, Edgar M. Faison, Gage O. Leighton, Hong Wang, David C. Williams Jr. and Robert Riehn is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Consistent AFM tip shape leading to reproducible results – NANOSENSORS PointProbe® Plus Screencast passes 1000 views mark

The screencast held by Head of R&D Thomas Sulzbach  on the  NANOSENSORS PointProbe® Plus Silicon AFM probe series with a consistent tip shape leading to more reproducible results has just passed the 1000 views mark.

Congratulations Thomas!

Screencasts on the PointProbe® Plus are also available in Japanese

and in Chinese:

also on youku http://v.youku.com/v_show/id_XNzMyMDg2MjQ4.html

Size‐Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures

Targeted drug delivery and precision medicine have now become a new paradigm in cancer therapy, with nanocarriers, pharmacologically active drugs could be directly delivered into target cancer cells to manage or reverse the course of disease. Nevertheless, the recent critical issue for drug delivery systems is lack of efficient drug delivery carriers.*

DNA nanostructures have attracted considerable attention as drug delivery carriers. However, the transmembrane kinetics of DNA nanostructures remains less explored.*

In the article “Size‐Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures” Xi Chen, Falin Tian, Min Li, Haijiao Xu, Mingjun Cai, Qian Li, Xiaolei Zuo, Hongda Wang, Xinghua Shi, Chunhai Fan, Huricha Baigude and Yuping Shan describe how they monitored the dynamic process of transporting single tetrahedral DNA nanostructures (TDNs) monitored in real time using a force-tracing technique based on atomic force microscopy.*

The authors used special NANOSENSORS™ PointProbe® Plus PPP-BSI AFM probes for the single-molecule force tracing.

The NANOSENSORS PPP-BSI AFM tips were modified in two steps as described in their article: First they were washed by Piranha solution (V(H2SO4):V(H2O2) = 3:1) for  1  h,  cleaned  with  ultrapure  water  twice  and  absolute  ethylacohol  once  then the AFM tips were dried by argon gas, and cleaned under O3 for 30 min to  remove  other  impurities.  After  cleaning,  the  AFM  tips  were  modified  with  3-aminopropyltriethoxysilane[17]  to  generate  amino  group,  it  was  convenient  for  linking  the  heterobifunctional  PEG  (MAL-PEG2000-SCM,  FW≈2000, SensoPathechnologies, Bozeman, MT 1 mg mL−1). After drying with  argon,  the  tips  were  immersed  in  a  mixture  of  100  ×  10−9m  TDNs,  50 μL  NH2OH-reagent  (500  ×  10−3m  NH2OH•HCl,  25  ×  10−3m  EDTA,  pH  7.5),  and  50  μL  buffer  A  (100  ×  10−3m  NaCl,  50  ×  10−3m  NaH2PO4, 1 × 10−3m EDTA, pH 7.5). After functionalization for 1 h, the AFM tips were washed with PBS for three times and stored at 4 °C .*


Figure 1 from “Size‐Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures” by Xi Chen et al:
Schematic diagram of the force tracing test. a) Scheme of assembling the TDNs, from left to right, ssDNA are strands A, B, C, and D (red, orange, green, and blue), respectively. The strand A stretch out a 5‐base ssDNA strand with sulfydryl group. The strand C is labeled with cyanine‐3 (Cy3) fluorophores (Cy3‐TDNs). b) The diagram of connecting TDNs to the AFM tip, the TDNs is attached on the AFM tip with heterobifunctional PEG linker. c) The working principle of constant position mode force tracing technique. d) A typical force tracing curve with force signal (blue arrow).

*Xi Chen, Falin Tian, Min Li, Haijiao Xu, Mingjun Cai, Qian Li, Xiaolei Zuo, Hongda Wang, Xinghua Shi, Chunhai Fan, Huricha Baigude, Yuping Shan
Size‐Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures
Global Challenges 2019, 1900075
DOI: https://doi.org/10.1002/gch2.201900075

Please follow this external link to read the full article: https://onlinelibrary.wiley.com/doi/full/10.1002/gch2.201900075

Open Access: The article “Size‐Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures” by Xi Chen, Falin Tian, Min Li, Haijiao Xu, Mingjun Cai, Qian Li, Xiaolei Zuo, Hongda Wang, Xinghua Shi, Chunhai Fan, Huricha Baigude and Yuping Shan is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.