Hot off the Press

Hot off the Press highlights recently published work for the benefit of our readers. Our contributors this month have focused on the action of RNA polymerase on DNA, new developments in magnetic resonance imaging and the use of amyloid fibrils as nanomaterials. New contributors are always welcome. If you are interested please contact molbiosyst@rsc.org for more information, we’d like to hear from you.

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RNA polymerase ‘scrunches’ DNA

Numerous studies have been done to determine what happens on a gene promoter during the initial events of transcription; however, puzzles still remain. It is known that the RNA polymerase will produce short 9–11 nucleotide transcripts (abortive initiation) before it escapes the promoter for productive elongation. Footprinting studies suggest that the RNA polymerase does not move on the promoter during abortive initiation. How do we resolve these seemingly contradictory results?

In a recent issue of Science, Strick, Ebright, and colleagues have used a method of single-molecule analysis to study what happens during abortive initiation. By affixing a promoter and coding region to a magnetic bead and monitoring the position of the bead the authors are able to measure the melting of dsDNA to ssDNA. They found that the RNA polymerase melted a longer region of DNA during abortive initiation. This supports a model of ‘scrunching’ where the RNA polymerase doesn't move relative to the promoter during abortive initiation but pulls a short section of downstream DNA towards itself to transcribe the DNA. Further, the authors were able to monitor DNA melting during full, productive transcription cycles and estimated all transcription events involve a period of scrunching and abortive initiation. This powerful technique of single-molecule analysis should be useful in studies of other important questions in transcription.

 

A. Revyakin, C. Liu, R. H. Ebright and T. R. Strick, Science, 314, 1139-1143.

Reviewed by: Chase Archer, University of Texas, Southwestern Medical Centre, Dallas.

Ultra-sensitive magnetic nanoparticles for magnetic resonance imaging

Nowadays, there is great interest in magnetic resonance imaging (MRI). This technique allows imaging inside the body of opaque organisms, and its noninvasive nature has been widely adapted in clinical medicine. However, its low signal sensitivity has been a major limitation for biological applications, particularly in the development of molecularly targeted MRI contrast agents.

To overcome this low sensitivity, Jea-Hyun Lee and co-workers have reported a series of metal-doped magnetism-engineered iron oxide (MEIO) nanoparicle MRI contrast agents of spinel MFe2O4 where M is the +2 cation of Mn, Fe, Co or Ni. Nanoparticles were coated with the ligand, 2,3-dimercaposuccinic acid (DMSA). The DMSA-coated nanoparticles they synthesized had high colloidal stability at a salt (NaCl) concentration of 250 mM, across a wide pH range (pH 6-10) and in serum. Among the various metal-doped MEIO nanoparticles, Mn-doped MnFe2O4 (MnMEIO) showed the strongest MR contrast effect with a relaxivity value (R2, which equals 1/T2) of 358 l⁻¹ mmol⁻¹ s, while the R2 value of cross-linked iron oxide nanoparticles (CLIO) was 62 l mmol⁻¹ s⁻¹. They conjugated MnMEIO nanoparticles with the cancer-targeting antibody Herceptin, which specifically binds to the HER2/neu marker overexpressed in breast and ovarian cancers. With the MnMEIO–Herceptin conjugates, the cell line with low level of HER/neu overexpression was detected with a noticeable MR contrast in T2-weighted MR images. Further, more in vivo MR imaging experiments with nude mice showed that the small implanted tumors (~50 mg) were clearly detected by intravenously injecting MnMEIO–Herceptin conjugates (Fig. 1). Thus, the MnMEIO-Herceptin conjugates was demonstrated to be a high sensitivity probe for cancer cell detection and in vivo imaging of small tumors. This study of magnetic nanoprobing methods could provide the basis of new reagents for the real-time visualization of various biological events, such as cell trafficking, cancer metastasis, cellular signaling and interactions at the molecular and cellular level.

Fig. 1 MR images of nude mice showing that the small implanted tumors (~50 mg) were clearly detected by intravenously injecting MnMEIO–Herceptin conjugates. Image reprinted with permission from Nature Medicine, 2007, 13(1), 95. Copyright Nature Publishing Group (2007).

 

Jae-Hyun Lee, Yong-Min Huh, Young-wook jun, Jung-wook Seo, Jung-tak Jang, Ho-Taek Song, Sungjun Kim, Eun-Jin Cho, Ho-Geun Yoon, Jin-Suck Suh and Jinwoo Cheon, Nat. Med., 2007, 13(1), 95.

Reviewed by: Kenjiro Hanaoka, University of Texas, Southwestern Medical Centre, Dallas.

Mechanical properties of individual amyloid fibrils as nano-materials

The formation of amyloid fibrils, insoluble filamentous aggregation of peptides and proteins, has been demonstrated to be a general property of every polypeptide, under suitable conditions. These structures are now well described, and many general structural features are maintained across very different proteins. Furthermore, the fact that the amyloid fibrils are reproducible self-assembled nano-structures makes the study of their nanoscale properties a very interesting field.

The idea of using amyloid fibrils as nano-materials led Prof. Christopher M. Dobson's group to join efforts with the Cambridge Nanoscience Centre, and Dr Mark E. Welland. They have determined the nanoscale properties of individual insulin amyloid fibrils, such as mechanical rigidity and resistance to breakage, through a clever AFM-based approach: amyloid fibrils were spread onto a gold-coated silicon surface to perform force microscopy; the surface had several grooves patterned on it, so that fibrils lie suspended over the grooves; by then applying force to the suspended fibrils, the cantilever probes the mechanical properties, strength and stiffness. Interestingly, two-filament insulin fibrils showed ultimate strength values of 0.6 ± 0.4 GPa, very similar to spider silk (1–1.5 GPa) or steel (0.6–1.8 GPa), with forces of 300-500 pN required to break the fibrils. The analysis of bending caused by thermal fluctuations confirmed the results from AFM. The mechanical properties described in the work are not only of interest in nano-science, but also give mechanistic insight into fibril formation. The breakage of fibrils enhances aggregation rates, since the number of fibril-ends available to grow increases, therefore this process is crucial to fibril growth.

In summary, the large strength and stiffness of amyloid fibrils explain on one hand the resistance to degradation and accumulation in tissues in diseases, and in the other hand open up future applications of fibrils as nano-materials.

 

Proc. Natl. Acad. Sci. U.S.A., 2006, 103 15806-15811

Reviewed by: Angel Orte, Department of Chemistry, University of Cambridge.

Hot off the RSC Press

Quantitative venom proteomics

Soluble dendrimers have been used to study differences in protein abundance in complex snake venoms.

Quantitative proteomics is used to detect differences in the protein profiles of cells or tissues in different states, and is becoming an increasingly important technique for biomarker and disease discovery. A common approach for this is to use a solid phase reagent to simultaneously capture and label proteins in solution for analysis.

Andy Tao and colleagues at Purdue University, West Lafayette, have developed a new strategy for quantitative proteomics called soluble polymer-based isotopic labelling (SoPIL). They used the technique to quantify differences in protein levels and enzyme activity between two different samples of snake venom. Snake venoms contain complex mixtures of proteins and other pharmacologically active molecules. According to Tao, they could be useful tools for drug discovery.

Tao's method uses a polymer, a soluble dendrimer, to bind and deliver an isotope label to proteins containing the amino acid cysteine within a solution mixture. An alkyne group on the dendrimer then acts as a handle to join the proteins to a solid phase reagent, allowing them to be removed easily from the solution. The tagged proteins can then be released from the solid and analysed by mass spectrometry. Compared to the established single-step solid phase capture and tagging method, the SoPIL two-step strategy produced labelled proteins much more quickly, and isolated the target protein in a higher yield.

Lingjun Li, an expert in mass-spectrometry-based protein analysis from the University of Wisconsin, Madison, US, explained that the SoPIL method could have many uses in proteomics research. By choosing different reactive handle groups, a wide range of applications, including probing various protein modifications in biological systems, should be possible, she said.

According to Tao, the soluble dendrimers also offer potential for in vivo proteomics. This would give a more accurate indication of the proteins in the living cell, as it is not known how physically disrupting cells for in vitro analysis affects their protein concentrations and distributions, he said.

 

M Guo, J Galan and WA Tao, Chem. Commun., 2007, DOI:10.1039/b614926j.

Reviewed by, Katherine Vickers, Royal Society of Chemistry, Cambridge.

Sickle cell microcell

A method to deoxygenate blood's oxygen-carrier, haemoglobin, could lead to treatments for sickle cell anaemia. London-based chemists have prepared a microcell that can be used to study the causes of the debilitating genetic condition.

In sickle cell disease, the patient's red blood cells are curved into a sickle shape and can become stuck in blood vessels, blocking blood flow and causing pain. Sickle cell haemoglobin (HbS) is different from normal haemoglobin (Hb) by just a single amino acid. This amino acid replacement means that HbS tends to polymerise into long insoluble fibres and it is this that changes the shape of the cell.

'The key to HbS polymerisation is the deoxygenated molecule, as fibre formation occurs only in this state,' said Daren Caruana, an electrochemist at University College London, UK. Caruana and colleagues have developed a novel technique for removing oxygen from HbS by electrochemical reduction allowing them to control and study HbS polymerisation in detail.

The group used the new technique to reduce a small volume of HbS within a custom-built transparent cell and were able to follow the resulting polymerisation as a change in turbidity.

The electrochemical method offers an advantage over previous deoxygenation methods, since these use oxygen scavengers which can have a negative effect on HbS structure, said Caruana.

‘Sickle cell disease is an international health problem affecting millions of people around the world,' said Caruana. ‘This method could not only be used to gain an understanding of the pathophysiology of sickle cell disease, but also as a screening method for drugs that could lead to novel therapeutic strategies for disrupting HbS polymerisation in patients.'

Fig. 2 Optical microscopy images showing HbS fibre growth. Frames d and e show a control. Figure reprinted with permission of the Royal Society of Chemistry.

 

Z Iqbal, R McKendry, M Horton and D J Caruana, Analyst, 2007, 132, 27.

Reviewed by Michael Spencelayh, Royal Society of Chemistry, Cambridge.

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