Universal DNA Sensor Boosts Potential for Carbon Nanotube Sequencing
Stuart Lindsay and his team at Arizona State University have developed the first DNA sensor that can discriminate between each of the four DNA bases on a single molecule of DNA and without the need for labels. This could be a step towards the highly anticipated era of US$1000 genome sequences and personalized diagnostics.
The advent of nanobiotechnology has led to several proposals for new methods for fast and inexpensive DNA sequencing. Lindsay has been working on the idea of DNA sequencing through nanopores. This is based upon the idea of passing a single strand of DNA through a pore, using an applied voltage, and measuring ion current or speed of translocation through the pore as a function of sequence.
The team first demonstrated the ability to read individual DNA bases, using four separate sensors, in 2008. The method used a β-cyclodextrin ring as a pore and atomic force microscopy (AFM) to measure minute changes in force, as the ring translocated along the DNA molecule. More recently, they demonstrated the ability to transport small single-stranded DNA oligomers through carbon nanotube pores with marked detectable transient increases in ion current.
In their latest innovation, Lindsay's group has employed modified gold electrodes. Attached to the end of AFM probes these electrodes act as ‘chemical tweezers’ which can hold the DNA. The gap of the tweezers has been optimized to 2.5 nm so that when a single chemical base of DNA passes through, it momentarily binds to the electrodes and an increase in current can be detected. This required careful fine-tuning of the apparatus. “What we did was to narrow the number of types of bound configurations to just one per DNA base,” Lindsay explains. “The beauty of the approach is that all the four bases just fit the 2.5 nm gap, so it is one size fits all, but only just so.”
The group hopes that the sensor could eventually be combined with their carbon nanotube technology to read the sequence of the DNA as it passes through the pore. An important step will also be to adapt the sensor to work in water-based solutions. If the process can be perfected, it will enable low-cost rapid DNA sequencing with important implications for personalized medicine and diagnostics.
Source: Liu H, He J, Tang J et al.: Translocation of single-stranded DNA through single-walled carbon nanotubes. Science 327(5961), 64–67 (2010);www.nanotech-now.com/news.cgi?story_id=36735; Ashcroft BA, Spadola Q, Qamar S: An AFM/rotaxane molecular reading head for sequence-dependent DNA structures. Small 4(8), 1468–1475 (2008); Chang S, Huang S, He J: Electronic signatures of all four DNA nucleosides in a tunneling gap. Nano Lett. 10(3), 1070–1075 (2010).
Nano-Sized Imaging of Cardiomyocytes
UK researchers have found cellular differences between the cardiac cells of healthy and of heart-failure rats using latest nanoscale imaging techniques
Scientists have reported signaling differences between the cardiac cells of a rat model of chronic heart failure and the cardiac cells of healthy rats. The differences lie in the spatial distribution of the β;2-adrenergic receptors (β2-ARs). The researchers, from Imperial College London (UK), found that in the cardiomyocytes of healthy adult rats, the β2-ARs are localized exclusively to the deep transverse tubules. However, in the rat model of chronic heart failure, the β2-ARs are situated on the cell crest. This difference in location of the β2-ARs has large implications for the cellular signaling pathways of the cardiomyocyte. In the chronic heart disease model, this redistribution led to diffuse receptor-mediated cAMP signaling. The authors hypothesize that this contributes to the failing myocardial phenotype.
Although β1-ARs and β2-ARs are known to mediate distinct effects on cardiac function, the effect of the spatial localization of β-ARs is less documented. The β-ARs regulate the production of the cAMP and are coupled to the G protein.
The researchers combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques to carry out the study protocol. The technique enables the surface of indivual cardiomyocytes to be imaged in unprecedented detail, providing the researchers with a unique insight into the components of the cell.
Author Julia Gorelik comments, “Our new technique means we can get a real insight into how individual cells are disrupted by heart failure. Using our nanoscale live-cell microscopy we can scan the surface of heart muscle cells to much greater accuracy than has been possible before and to see tiny structures that affect how the cells function.”
It is hoped that the findings will lead to improvements in therapeutics for heart failure and abnormal heart rhythms. In particular, the research may have implications for the design of β-blockers. Gorelik explains, “Through understanding what's happening on this tiny scale, we can ultimately build up a really detailed picture of what's happening to the heart during heart failure and long term, this should help us to tackle the disease.”
Source: Nikolaev VO, Moshkov A, Lyon AR et al.: β2-adrenergic receptor redistribution in heart failure changes camp compartmentation. Science (2010) (Epub ahead of print).
Nanoparticles Coated with Activatable Cell Penetrating Peptides Are Useful in High-Resolution Imaging, Study Suggests
Molecules intrinsically involved in malignancy and metastasis can now be studied in vivo by MRI and fluorescence of dendrimeric nanoparticles coated with activatable cell penetrating peptides (ACPPs) that have been labeled with Cy5, gadolinium or both.
Olson and colleagues from the Howard Hughes Medical Institute, University of California at San Diego (CA, USA) have successfully modified ACPPs for use in both MRI and fluorescence imaging.
Improvement high-resolution imaging allowing visualization of matrix metalloproteinase activity will be valuable for the clinical detection and staging of tumors.
Attaching a large molecular weight to the ACPP improves contrast between tumor cells and adjacent healthy tissue cells. The additional molecule bound to ACPP lowers teh rate of background uptake into healthy tissues. This leads to a greater difference between the healthy and cancerous cells.
The modified ACPPs also allow a greater amount of contrast agent to be deposited in the tumor cells. The researchers found that by attaching the molecular weight to the polycationic domain of the ACPP it becomes part of the cargo, allowing multiple contrast agents to be carried.
The amenability of ACPPs for duel labeling for magnietic resonance imaging and fluorescence gives them an advantage over other optical and MR-based probes. Another advantage is that the uptake of those ACPP nanoparticles in tumors is approximately 4- to 15-fold higher than of unconjugated ACPPs.
ACPPs are also useful in that they are activated by proteases that are upregulated by a variety of tumors from around the body. This means that they show more promise as diagnostic agents than molecules that target particular antigens only upregulated in certain cancer cells.
The duel label promises to benefit the future of fluorescence-guided surgery. The use of fluorescent molecules allows detection of residual tumor and metastases just 200 muM in size. The Gd-labelled nanoparticles can be activated to deposit high levels (30–50 muM) of Gd in the tumor cells, resulting in useful T(1) contrast, which lasts for several days after injection.
As well as fluorescence-guided surgery it is hoped that the advances made by this study will improve MRI-guided staging and presurgical planning. The approach may also be used to selectively deliver radiation-sensitizing and chemotherapeutic agents.
Source: Olson ES, Jiang T, Aguilera TA et al.: Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imagig of proteases. Proc. Natl Acad. Sci. USA 107 (9) 4311–4316 (2010).
Designer Nano Containers Created to Deliver Drugs to Diseased Cells
Empty virus-like particles can finally be loaded with useful chemicals, bringing us one step closer to targeted drug delivery
Scientists from the John Innes Centre, UK, have succeeded in growing cowpea mosaic virus particles, emptying them of their genetic material and then loading them with therapeutic agents.
The idea is to design nanoscale drug containers that can be targeted to specific sites within the body by attaching guiding molecules to the external surface of the virus. Upon reaching the target site the virus-like particles would then discharge their chemical load.
The step enabling scientists to decorate the surface of the cowpea mosaic virus with targeting molecules has been previously achieved, but until now scientists have been unable to sufficiently empty the virus particles in order to be able to fill them with the desired chemicals.
“Now we can load them too, creating fancy chemical containers,” explains Dave Evans, John Innes Centre, UK, lead author of the paper soon to be published in Small.
To turn the cowpea mosaic virus particle into virus-like drug containers the particles must be emptied. RNA needs to be removed from the particles in order to render them noninfectious. This stage of the procedure has been addressed.
“This is a shot in the arm of all cowpea mosaic virus technology,” comments George Lomonosoff.
The cowpea mosaic virus is an ideal candidate to use when designing biomaterial at the nanoscale. The virus can be readily isolated from plants and the genetic, biological and physical properties have been well characterized.
“This brings a huge change to the whole technology and opens up new areas of research,” Lomonosoff elaborates. “We don't really know all the potential applications yet because such particles have not been available before. There is no history of them.”
It is hoped that the new technology will enable more targeted drug delivery. This would be particularly useful for the delivery of cancer drugs, which can be known to damage healthy cells along with the cancer cells, leading to undesirable side effects such as hair loss.
The virus-like particles could be decorated with peptides that bind to integrins, which are molecules that appear on cancerous cells. This would allow the particles to be directed to cancer cells rather than healthy cells. Once bound to the intended cancer cells the particles would release their cargo – the anti-cancer drug.
“The potential for developing Cowpea mosaic virus as a targeted delivery agent of therapeutics is now a reality,” says Evans.
The researchers are filing for a patent to protect the technology – the empty virus-like particles, their use and the process carried out to produce them. The patent filing and commercialization of the new technology is being managed by PBL.
Source: Aljabali AAA, Sainsbury F, Lomonosoff GP, Evans DJ: Cowpea mosaic virus unmodified empty virus-like particles can be loaded with metal and metal oxide. Small DOI: 10.1002/smll.200902135 (2010) (Epub ahead of print).