Live and Let Dye

Live cell imaging of RNA dynamics traditionally relies upon microinjection of fluorescently tagged RNA molecules, strategies involving coexpression of a fusion between a fluorescent protein and an RNA binding protein, or variants of FISH. While these approaches are ideal for localization of specific transcripts, they clearly are less suited for investigating overall RNA dynamics. Li et al., explain that a global analysis of RNA behavior in the nucleus requires a cell-permeant fluorescent dye that selectively binds RNA while ignoring DNA. When extant offerings proved unsatisfactory, the authors used their expertise in chemical boilogy to generate new tools. First, Li et al., prepared a styryl dye library, a strategy based on their previous work, demonstrating that these dyes are compatible with live cell imaging and can be persuaded to bind well to nucleic acids (although their previous efforts focused on DNA). Two rounds of screening, which included examining changes in the candidate dyes’ fluorescent emission intensity upon binding RNA in solution and analysis by live cell imaging, produced three RNA-responsive dyes meriting further characterization. One property of immediate concern was the cytotoxicity of these new agents. MTS assays revealed that significant cytotoxicity became apparent only after prolonged incu- bations (>24 h) at the 5 µM working concentration. UV-induced toxic- ity proved comparable to Hoechst staining. The dyes also shine with promise when it comes to more demanding cell staining applications. Although one of the two dyes visible in the FITC channel (the other styryl dye requires a Cy™3 filter) showed inconclusive results in DNase and RNase tests in fixed cells, the other compounds behaved with the expected RNA selectivity. Moreover, all three dyes are compatible with counterstaining by Hoechst or DAPI, a critical feature for investigations of nuclear organization. The authors are making the photostable, RNA- selective styryl dyes available to investigators interested in collaborative research. –ND

- Li et al. 2006. RNA-selective, live cell imaging probes for studying nuclear structure and function. Chemistry & Biology 13:615–623.

Bring It On

In another superb example of cross-disciplinary research cooperation, chemistry and biochemistry researchers have together moved a large step—possibly even a leap—closer to highly targeted disease diagnosis and treatment. Monoclonal antibodies have now come of age as an effective cancer therapy, predominantly due to their exquisite specificity and relative low toxicity. In a new variation on this theme, the authors have developed chemically programmed antibodies (cpAbs), which are conjugates of antibodies and small molecules or peptides. Covalent modification of a specialized catalytically active monoclonal antibody (MAb) using a specially designed adapter molecule results in reprogramming of the antibody so that it acquires the specificity of the attached small molecule. Then, in a process analogous to a precision military missile strike (but with significantly less collateral damage) the cpAb is used to target disease cells and destroy them. In this scenario, the small molecule or peptide provides the specificity (e.g., acting as a ligand for a particularly cellular receptor), while the antibody provides stability and extended halflife in vivo. The creation of cpAb conjugates has been described previously by the authors, who now extend the work using a new proadapter that undergoes a two step process; in the first step a vinyl ketone reactive moiety is exposed, which then drives the reaction to covalently join the antibody to the small molecule in the second step. The key improvement in this system over the previous single-step, highly-active diketone model is that the antibody catalyses the first step of activating the relatively inert proadapter, thereby effectively making its own adapter. This system gives researchers and clinicians more flexibility in treatment regimens through in vivo formation of cpAbs by injection of the MAb and small molecule separately or by in vitro complex formation with subsequent injection. –SS

– Guo et al. 2006. Breaking the one antibody–one target axiom. Proceedings of the National Academy of Sciences 103(29):11009–11014.

An Affinity for Methylation Detection

Methylation of CpG dinucleotides located in eukaryotic promoter regions is now well known to play an integral role in promoter function and thereby the regulation of gene expression. Many cellular pathways, from developmental control to apoptosis and ageing, as well as dysfunctions such as cancer and other diseases, have been shown to be influenced by differential methylation of essential genes. The majority of methods currently used to detect methylation patterns involve methylation sensitive restriction digestion or bisulfite treatment,usually followed by PCR amplification—few alternative techniques have been successfully proposed or adopted. Now, however, Gebhard et al., present an elegant and speedy new methylation detection system that works independently of any DNA modifying treatments, apart from prefragmentation of the genomic DNA byrestriction digestion to obtain manageable sized targets. Their so-called methyl-binding PCR (MB-PCR) utilizes a recombinant protein with a high affinity for methylated CpG sequences to bind and retain relevant sequences, which can then be detected by gene-specific PCR in the same tube. By adjusting the salt concentration in the binding buffer, the specificity of CpG binding can be modulated. MB-PCR provides a more global indication of the methylation status of a particular region rather than drilling down to individual CpG islands as is done with methylation-specific PCRs or sequencing following bisulfite treatment. The method can detect the level of methylation (high, intermediate, or none), which the authors claim is adequate to identify hypermethylation in tumor samples. To demonstrate this, MB-PCR was used to confirm the methylation status of known methylated and unmethylated genes from cell lines, as well as to identify a putative target for methylation in leukemia cell lines and certain patients with acute myelogenous leukemia. –SS

- Gebhard etal. 2006. Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR. Nucleic Acids Research 34(11):e82.

Ties That Bind

Understanding interactions between cell-surface receptors and ligands is relevant to research deciphering the processes underlying such hot areas such as angiogenesis and stem cell self-renewal. An array format in which multiple potential ligands can be spotted and simultaneously assayed is clearly an attractive strategy; accordingly, a number of groups have described various protein, peptide, and small molecule arrays. Typically, cell attachment is the readout in such assays; however, it can sometimes be useful to distinguish an initial binding event from the subsequent spreading and flattening that make up cell attachment. In their recent study, Peelen et al., describe a cell capture assay that facilitates observation of defined interactions between a substate-bound ligand and cell-surface molecules in which the readout is cell binding, not attachment. The authors observed binding by either phase contrast microscopy or surface plasmon resonance imaging. Although the chemistry used in depositing the protein ligands of interest varied depending on the imaging modality, in each case the arrayed proteins were deposited on a gold thin film—a substrate also applicable for fluorescence micros- copy and MALDI mass spectrom- etry. In the Peelen et al., study, SPR imaging proved a convenient, sensitive, and reproducible method for assessing binding. However, its spatial resolution is not refined enough to distinguish individual cells. Therefore, the authors used conventional bright field microscopy to assess bona fide cell attachment following a 2-day incubation period. When interactions between BHK-21 cells and basic fibroblast growth factor (bFGF) were examined, the results supported the conclusion that there was specific cell binding but the absence of attachment. The authors propose that the defined chemical composition of each arrayed spot explains the discrepancy with previous literature and allows more detailed analysis of the ligands that are both necessary and sufficient for cell attachment. – ND

- Peelen et al. 2006. Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films. Journal of Proteome Research 5:1580–1585.