DNA Nanotechnology - Science topic

Dear jiahoa

I recumend this document for you

Hi, I hope you already found an answer to this, but I think for cubes and other structures with specific dimensions ADENITA is a good option. Adenita SAMSON edition is a SAMSON (https://www.samson-connect.net/) plugin. you only need to add the app in the shop. You can use freeware option of SAMSON and it works quite nice.

De Llano, E.; Miao, H.; Ahmadi, Y.; Wilson, A.J.; Beeby, M.; Viola, I.; Barisic, I. Adenita: interactive 3D modelling and visualization of DNA nanostructures. Nucleic Acids Res. 2020, 48, 8269–8275, doi:10.1093/nar/gkaa593.

Best

Nanopores have emerged as versatile sensors for detection and analysis of single

molecules and particles over the past decade [1-7]. Similar to Coulter counters, nanopores

employ resistive pulse sensing in which ionic current through the pore changes during

translocation (passage) of a molecule through the pore [2]. Particularly, attention has

been focused on developing nanopores for rapid sequencing of DNA using α-hemolysin

and solid-state nanopores [1]. While close interactions between the nanopore and the

DNA molecule can yield structural information about the molecule, larger nanopores in

the 10-500 nm size range are better suited for analysis of physicochemical characteristics.

Saleh and Sohn [4] used rapid prototyping in PDMS (polydimethylsiloxane) to fabricate

200-400 nm nanopores for detection of λ-DNA, while Fan et al. [5] used silica nanotubes

for sensing λ-DNA. While larger nanopores are relatively easy to fabricate, the signal-to-

noise ratio is compromised due to the large size of the nanopore. To address this issue,

we are developing devices to perform multiple measurements on long DNA molecules,

which could significantly improve the signal-to-noise ratio by statistical averaging on the

same molecule [8, 9].

Knowledge of the factors that affect the frequency of translocation events is critical

for designing such nanopore devices and for understanding the mechanisms that govern

transport of these molecules through the nanopore. Study of DNA translocation through

α-hemolysin nanopores [10, 11] and solid-state nanopores [12] has yielded valuable

insights into the effect of voltage bias, DNA concentration, DNA length, etc. on the

translocation frequency, duration, and current blockage. However, relatively little is

known about translocation of long DNA molecules through larger nanopores that involve

a significantly different pore geometry, size, and electric fields. In this paper, we examine

the effects of voltage bias and DNA concentration on the translocation of λ-DNA through

PDMS nanopores.

Nanoparticle-Based Strategies to Combat COVID-19

...Nanotechnology has tremendous applications in the food, pesticide, fertilizer, chemical, and agriculture industries. Nanostructures or nanoformulations are fabricated by manipulating, at atomic or molecular level, reactants in definite ratios for improving the physical, chemical, and conduction properties as well as strengthening the functioning materials applicable in agriculture, medicine, and environmental monitoring. ... The nanoparticles, APC molded into functional nano-biopesticides via green technology, could selectively target insects for plant and environmental safety. ... Lade, B. D., & Gogle, D. P. (2019). Nano-biopesticides: Synthesis and Applications in Plant Safety. In Nanobiotechnology Applications in Plant Protection (pp. 169-189). Springer, Cham.

Hi Logan,

Since you already have interested suppliers, why not contact their customer service for quotations? Different companies have different technical support services and final delivery quality, so it’s difficult to compare, depending on whether you care more about data quality or cost.

Dear Hammond,

The oligo am using is Thiol C6-Modified, The electrodes are so small that, it can be dipped in 96 Well plates. Am adding 150ul of Oligo's of different concentrations to the well (the volume is made such that the working electrode has to be completely submerged) and dipped the electrode for 180 mins at 30^o C. Washed with buffer to remove any unbound oligo's and treated with 6-Mercapto-1-hexanol (MCH) for an hour at 30^o C, further washed with buffer to remove any unbound MCH and electrochemical studies are carried out.

What protocol are you using? If you're having issues with the binding of the proteins to the nanoparticles it means your nanoparticles are problematic. Try using a nanoparticle reagent with cationic elements to ensure that you get through the cell membrane (see https://altogen.com/product/nanoparticle-in-vivo-transfection-reagent/). That should help improve your results, and once again, be sure to follow protocols.

MeshLab? 

If the particles are stable in an aqueous buffer you can follow the procedure described by Hermanson (Bioconjugate Techniques 2nd ed) in Chapter 24. 

Divalent cations have been used to deposit DNA origami on silicon oxide, but the concentration required is above 100 mM Mg2+ and the surface coverage is low. (See Kershner et al, Nature Nanotechnology, 2009, 4, 557-561 and Albrechts et al. "Adsorption studies of DNA origami on silicon dioxide." In: 21st Micromechanics and Micro Systems Europe Workshop, MME 2010).

A more reliable way that I use to deposit DNA origami on silicon (with native oxide in my case) is through APTES functionalization. The APTES monolayer is covalently bound to the oxide and the terminal amine is protonated in DNA origami buffer conditions. This provides the positive charge for the electrostatic interaction with the negatively charged DNA origami backbone. I have used 2D origami as well as DNA "nanotubes" with APTES on silicon with good results. However, you may find that it is the spherical nature of your nanostructures that is making it difficult to image. There are well established protocols for SiO2 APTES functionalization (only a few resources: Sarveswaran et al. Proc. of SPIE-Int. Soc. Opt. Eng, 2010, 7637, No. 7637M; Kim et al. Soft Matter, 2011, 7, 4636-4643; Pillers et al. Journal of Visualized Experiments, 2015, 101, e52972).

Are you imaging in liquid or in air? If you are imaging in liquid, "anchoring" the DNA origami with the methods described above is not feasible because, as you said, the DNA origami is mobile. If in air, are you using tapping/non contact mode? This is essential to ensure that the fragile DNA origami is not ruined by the AFM imaging (contact mode imaging will tear the nanostructures).

Feel free to directly message me if you have any other questions.

You have to check first if the assembly has occurred or not. Some of nanoparticles might be not conjugated to the protein. I suggest making the assembly according to one of the reproducible protocols in the literature. Thereafter, use electrophoresis to separate the assemblies, take some samples and do TEM or SEM ( based on the particle size you expect). You might try Cryo-TEM if your particles are small, which I think can help you a lot.   

Both should work provided your conjugation strategy (reaction condition!) does not impede the activity of siRNA; as well as it releases the siRNA well under desired target site.

Circular dichroic (CD) spectroscopy

Hi, 

Yes, if you use a sample from a glycerol gradient directly for TEM, I suspect the glycerol can  affect your TEM samples. When I have used glycerol gradient centrifugation I have removed the glycerol by dialysis or spin filtration before TEM imaging.  Glycerol gradient centrifugation may not be the most simple way to remove staples from an origami sample, for simple TEM studies agarose gel extraction should be enough. 

Have you tried TEM on the sample without removing the staples? This is should be no problems. 

As for the incubation times in the TEM sample prepartaion, we normally only leave our samples on the grid for 20 seconds and only stain in uranyl formate for 20s. 

It may be helpful if you share the protocol you are using now so we can comment on it. 

Erik 

Hi Tiago,

From your question, I guess that you want to link unmodified DNA with a nanoparticle in a way that ensures that your structure is not disturbed.

I'm not sure if this is feasible. You could extend one of the strands of your origami and have a freely floating sticky end in solution. You could then take magnetic nanoparticles with the complement of the sticky end attached to it. This could be done by using thiolated DNA and gold nanoparticles. The sticky ends on the nanoparticle and the origami should hybridize, connecting both.

This issue is complicated by the size of the origami and the nano-particles; whether they are too big to be effectively connected by dsDNA. I'm aware that sticky ended cohesion can be used to link gold nanoparticles together. Another issue is that this interaction is not permamanent.

On a related note, you could extend one of the strands in the origami and place a thiolated base at the end of the same.

Regards,

Atul

Method 2: requires only the number of water molecules in the box.

N_ions = 0.0187 x ionConc(M) x no. of Water molecules

This discussion related to this is available in VMD forum n the link is provided here.

Several possibilities.  Without additional details given specifics are hard to sort out but:

Hope this helps...

Hello,

You absolutely right. Your final DNA and drug concentrations will change in this case. You will need increase concentration of DNA and drug on the factor of dilution. For exp. if you will dilute to 1:1, you will need take of 2 fold large of concentration of both (DNA and drug).

Our team produced an article, where an easy process is fully described.

DOI: 10.1021/acsami.5b01191

Dear Kan,

RNA and DNA aptamers against lysozyme have been taken to town on their biophysics. The paper I unsuccessfully tried to attach (Potty et al., DOI: 10.1016/j.ijbiomac.2010.12.007) has a fairly clear discussion on the contribution of each thermodynamic parameter.

Best wishes,

Vitor

I hope this paper can help you

W. Haiss et al., Determination of Size and Concentration of Gold Nanoparticles from UV-VIS Spectra,

Anal. Chem. 2007, 79, 4215-4221

well, if you know the DNA conc. and also AuNP conc. then you just need to divide DNA conc. over AuNP. You will get how many DNAs are on the gold nano particles. Let me give you an example: if your thiol-DNA conc is 2 micro M and Gold conc. is 13 nM then 2000 nM/13 nM= 153 DNA per gold nano particles. For your case, it looks you have 160 DNA per nano particles at high coverage, and 80 DNA per gold nano particles at low coverage.

Thank you sir for your pointers.That would definitely lead me to right direction. I will read and try to modify and do experiment as per your suggestions.Will get back to you for sure. :)

PEG can help reduce protein adsorption. There is commercial lipoic acid-PEG-maleimide molecule you can purchase to still be able to use thiolated DNA on top of the PEG coating. However, I would always expect a protein corona around DNA coated particles, irrespective of the specific DNA sequence. There is a lot of work on protein corona out there, just search for it.

You can go for MFold version 3.6 available at http://mfold.rna.albany.edu/?q=mfold/download-mfold

As far as you have for example poly-A end of your DNA, you can follow some protocols. Otherwise you can modify AuNPs with some short thiolated DNA, partially complmentary to your DNA and then bind your DNA.

Attaching DNA to AuNP without any treatment is not so effective. You will only achieve non stable DNA@AuNPs because of the nonspecific interactions. DNA without thiol binds to gold via backbone or N+ from the bases, but these bonds are realy weak.

While clustering of small nanoparticles should in theory increase the emission intensity, at least in the sense that the local concentration of emitters will be higher, it may not be quite this simple.  For one thing, are you planning on using core or core-shell Quantum Dots? I ask as clustering of particles may result in quenching of particles if they are too close due to electron transfer between adjacent particles, especially if you are using core particles.  At the very least the interparticle separation may prove important.

Another consideration, depending on what environment the particles will be in, is that a small cluster of particles that have the same 'footprint' on a 2D substrate will have a lower volume and a greater surface area.  While the lower volume may not be critical, the higher surface area means they will be more easily quenched by surface effects.  

Lastly, if you are simply interested in having high intensity green emission, it might be better to use one of the recent approaches to particle synthesis that employ either 'giant shells' of CdS on a CdSe core, or alloying of CdSe/CdS/ZnS.

its a very slow process, but the pH must maintain at 8 to 9 and keep the sample for a while.

Hello

I have worked on DNA origami platform. I have fabricate DNA logic gates as well as nano materials to DNA origami. I have experience to characterise this assembly by Atomic force microscope and alternative methods. If you need help in this regard, let me know.

Amit

Surface passivation of MWCNT in conjugation with organic molecules result fluorescence. Similarly DWNT by interaction with single stranded DNA shows fluorescence. One can use different potential molecules for similar surface passivation to invoke fluorescence and can tune the intensity of the fluorescence or even can change the emission energy. In contrast carbon dots, onions or graphene simply on oxidation result peripheral hydroxylation and/or carboxylation in carbon dots or carbon onions to fluoresce in aqueous solution. Similarly oxidation of graphene to graphene oxide which can be readily done through its chemically oxidative synthetic procedure in aqueous medium as precursor of graphene shows fluorescence. I stress aqueous medium because in biological investigation that is the best medium

In many cases, this will inhibit exonuclease activity. For example, ExoI is blocked by 3' phosphoryl or acetyl groups (http://www.jbc.org/content/239/8/2628.long).

For ExoI, I would assume that this holds true for most other modifications.

The surface coverage will depend on the radius of the NPs as well as its charge. The surface charge density will depend on the ionic strength and the pH of the solution. But basically all protein will adsorb to gold, the time of residence is just not the same. If your protein has a cystein group , you can expect a stronger affinity via covalent bond ( thiol bonding). DNA is the most charged polymer in nature so you can expect strong electrostatic interaction with the DNA lying down onto the NPs surface

Hi, i use NUPARM/NUCGEN and "nanoengineer" to device small DNA designs like tetra, icoshedron cages, as well bundles of DNA tubes. i can let you know the details if this what u r looking for

HI Himanshu,

You should have called me over phone or dropped a mail. this is my bread and butter. :)

Good that i saw it in RG, some more points to mr RGScore :)

Ok, coming to your question.

DNA is made of four bases A,T,G and C. and A pairs with T and G pairs with C. a sequence of DNA which forms a duplex will have one base pair stacked over the other. The paper talks about wat is known as "dinucleotide steps.". Its nothing but the combination of base pair stacked. a total of 16 possible combination can occur. Take "AA/TT" as a example. It refers to stacking of Adenine bases in strand_I and Thymine base in strand_II. Ur question is why is that they have not calculate the energy for TT/AA. "TT/AA " has thymine in strand_I and Adenine in strand_II. if u pass a pseudo-symmetry axis in the center of the step. u can go btw AA/TT and TT/AA.

I suggest u take coordinates of these two steps . superpose n observe that 5' to 3' direction is maintained even after 180 degree rotation.

Thus, Instead of 16 steps there is only 10 possible unique combination of steps.

Hope this helps.