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n e w s ANALYTICAL CURRENTS Low-affinity complexes How good is electrospray ionization MS stants for ligand-to-complex ratios rang - lieved to be hydrogen bound to the (ESI-MS) at detecting weakly bound ing from 1:1 to 4:1 were determined. RNA. Finally, this tour de force study complexes? Richard Griffey and col - However, the chemistry is even more reports the relative gas-phase activation leagues at Ibis Therapeutics report a complex, because 2-DOS binds to two energies for the dissociation of a series spectrum of a complex held together distinct sites on 16S. To investigate the of ligand–16S complexes. ( J. Am. Chem. by a single hydrogen bond. They also stability of these two forms, the re - Soc. 2000, 122, 9933–9938) describe the binding stoichiometry for searchers turned to colli - complexes of multiple ligands with RNA sionally activated dissocia - and an example where a ligand binds to tion and MS/MS. By 16S [M-5H+]5- two different sites. The key is choosing varying the relative disso - the right conditions for the ESI-MS ciation energy, the com - experiment. plex was dissociated into The experiments focus on low-affinity the 1:1 complex and the complexes formed with a 27-nucleotide free 16S and 2-DOS ions. 16S + 2-DOS RNA model of the 16S rRNA A-site MS also revealed multi - (16S). As a first step, the researchers ad - ple different ligands con - 16S + DT just the “harshness” of ESI-MS condi - currently binding 16S. 16S + 2-DOS + DT tions by looking for the spectrum of One particularly complex ammonia-adducted ions of 16S. This experiment found the 16S + (2-DOS)5 spectrum appears by either lowering mono- and bis-ligand the capillary–skimmer potential or the complexes of 2-DOS and desolvation capillary temperature. 16S, plus a complex with 1720 1740 1760 1780 m/z Binding of the ligand 2-deoxystrept - 2-DOS and a diaminotria - Low-affinity complexes galore. ESI-FT ion cyclotron reso - amine (2-DOS) was the basis of several zole simultaneously bound nance mass spectrum showing the concurrent binding of experiments. In one, dissociation con - to 16S. The triazole is be - 2-DOS and 3,5-diaminotriazole to 16S. Dynamics with a tracer Here is a handy trick. David Padowitz and Ben - monolayer. However, they use scanning tun - central methylene group in the carbon chain jamin Messmore of Amherst College investi - neling microscopy (STM), which is too slow to with either a sulfur (thioether) or oxygen gate exchange between long-chain molecules follow the dynamics of individual molecules. To (ether), are mixed with the alkane or with in solution and those arranged as a surface get around this problem, they introduce a trac - each other in a ~1:10 ratio. The mixture co- er molecule that allows the exchange to be fol - crystallizes on the graphite surface, and the lowed by the “slow-moving” STM. heteroatom is easily viewed. The system under investigation is the C 33 H68 The researchers find that the sulfur is par - molecule n-tritriacontane, which is adsorbed ticularly easy to track, and they can watch onto graphite. These molecules move on and the entire monolayer turn over in a few tens off the surface in milliseconds—much faster of seconds. The rate of exchange is deter - than the seconds required for an STM image. mined from the data, and the researchers Follow the bright dots. Two images tracking Instead of looking at individual molecules, speculate on the mechanism. They also see the thioethers on the surface. It takes 2 sec - onds to collect an STM image with a 2-sec - the researchers tracked the entire surface. different dynamics at the domain boundaries. ond delay between scans. To do that, tracer molecules, which replace a (J. Phys. Chem. B 2000, 43, 9943–9946) JANUARY 1, 2001 / ANALYTICAL CHEMISTR Y 9 A n e w s ANALYTICAL CURRENTS A rainbow of molecular beacons With the introduction of “wavelength- monochromatic light source. open, and, thus, the quencher and fluo - shifting” molecular beacons, the future Like traditional molecular beacons, rophore are apart. for these fluorescent probes looks espe - the wavelength-shifting version is a sin - However, there are two fluorophores cially bright, not to mention colorful. gle-stranded, hairpin-shaped oligonu - in the new molecular beacons—a “har - Sanjay Tyagi, Salvatore Marras, and cleotide probe that does not emit a sig - vester” that absorbs energy from the ex - Fred Kramer at the Public Health Re - nal when the hairpin is closed—that is, citation light and an “emitter” that re - search Institute in New York developed when the probe’s fluorophore and leases energy as a fluorescent signal. The the new probes, which emit light at var - quencher are close together. The signal emitter fluorophore is joined to the 5´- ious colors yet are excited by a single, becomes detectable when the hairpin is end of the probe by a short spacer se - quence, the length of which can be adjusted to optimize the fluorescence res onance energy transfer between the Measuring ligand–receptor forces two moieties. The researchers tested three wave - Cell–cell adhesion generally depends ducing receptor–ligand pairs. Depend- length-shifting molecular beacons. Each on specific binding between recep - ing on the force applied, the ruptur - one used fluorescein as the harvester; tors on the surface of one cell and ing of single or multiple receptor–li- the emitter was either 6-carboxyrhoda - ligands on the surface of another. gand bonds can be studied. mine 6G, tetramethylrhodamine, or The amounts of force needed to The researchers tested the setup Texas red. In all cases, the signal was break apart these receptor–ligand using the integrin receptor ␣IIb 3 detected predominantly in the emission pairs are typically measured “in bulk” from blood platelets and artificial li- range of the emitter, not the harvester. using macroscopic methods or nano - gands and found that the unbinding In addition, the researchers note that probe techniques at the single mole - forces were much smaller than those the new molecular beacons were often cule level. To help develop a more measured in single molecule studies. brighter than conventional ones, and complete picture, E. Sackmann and The reason for the difference, the re - they attribute that change to more effi - colleagues at the University of Cali - searchers suggest, is that the new cient energy absorption. ( Nat. Biotech - fornia–Los Angeles, the MPI for Bio - method seems to measure leverage nol. 2000, 18, 1191–1196) chemistry (Germany), and the Tech - forces, which may break the recep - nische Universität München (Germany) tor–ligand bonds efficiently, whereas describe a middle-ground “mesoscop - other methods measure pure traction Cystic fibrosis diagnostic ic ” method for measuring lig - forces. ( Langmuir, 2000, 16, 8984– array and–receptor unbinding forces. 8993) In the new method, a “test cell”— Although some mutations that cause cystic in reality, an artificially formed giant fibrosis can be identified by genetic testing, B- vesicle—is immobilized on a substrate field the more universal diagnostic technique is via receptor–ligand binding. Then a N S magnetic bead is attached to the top the so-called sweat test, or more correctly, of the cell, and a vertical force of 0.1–2 pN is generated by pulling the pilocarpine iontophoresis test for elec - on the bead. Reflection interference θF trolytes in sweat. Although the sweat test is contrast microscopy monitors the c convenient and simple to administer, analyz - structure, and the magnitude and di - R rection of the forces are determined c θO ing the samples is not as easy. But Aogán c quantitatively. h Lynch, Dermot Diamond, and Matt Leader at The approach allows the measure - ment of unbinding forces under bio - Dublin City University (Ireland) and SENDX logically relevant conditions, the re - Medical, Inc., hope to change that with a searchers say. Because the cell is large , A “test cell” is held to a substrate by re - the “pinning center” holding it to ceptor–ligand binding. A magnetic bead potentiometric ion-selective microelectrode the substrate may be ~1000 Å across attached to the top of the cell allows a array that may replace techniques such as and may contain ~100 adhesion-pro - vertical force to be applied. 1 0 A ANALYTICAL CHEMISTR Y / JANUARY 1, 2001 n e w s flame photometry and anion exchange chro - a. matography. DNA detects lead cleavage site substrate strand 17DS The array used is produced commercially 3'–GTAGAGAAGGrATATCACTCA–5' Decades of genetic research are pay ing 5'–CATCTCTTCT ATAGTGAGT–3' C A and consists of solid-state microelectrodes off in unexpected ways. In this case, C A G A G T G a deoxyribozyme, which is a catalyti - C G C enzyme strand 17E screen-printed over a salt-doped hydrogel cally active DNA, is the basis of a se - G C 2+ b. 80000 lective Pb biosensor. According to III layer. To give a more comprehensive analysis, I Yi Lu and Jing Li of the University +Enz the array simultaneously measures levels of y t 60000 I of Illinois at Urbana–Champaign, this i II s + – n e 2+ Na and Cl , which are the two most reliable in - unique sensor boasts a >80-fold pref - t +Pb n i 2+ 40000 + erence for Pb over other common e c dicators; K , which is often used to confirm the n III metal ions and a quantifiable detection e c 2+ s II e 20000 diagnosis; and other parameters, such as Ca , range of 10 nM–4 µM. Moreover, this r o u l approach suggests that a wealth of f pH, pO , and conductivity.