抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

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Principles Of Fluorescence Spectroscopy

Fluorescent labeling of biomolecules with organic probes.

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

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Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

DNA charge transport (CT) chemistry has received considerable attention by scientific researchers over the past 15 years since our first provocative publication on long range CT in a DNA assembly.1,2 This interest, shared by physicists, chemists and biologists, reflects the potential of DNA CT to provide a sensitive route for signaling, whether in the construction of nanoscale biosensors or as an enzymatic tool to detect damage in the genome. Research into DNA CT chemistry began as a quest to determine whether the DNA double helix, a macromolecular assembly in solution with π-stacked base pairs, might share conductive characteristics with π-stacked solids. Physicists carried out sophisticated experiments to measure the conductivity of DNA samples, but the means to connect discrete DNA assemblies into the devices to gauge conductivity varied, as did the conditions under which conductivities were determined. Chemists constructed DNA assemblies to measure hole and electron transport in solution using a variety of hole and electron donors. Here, too, DNA CT was seen to depend upon the connections, or coupling, between donors and the DNA base pair stack. Importantly, these experiments have resolved the debate over whether DNA CT is possible. Moreover these studies have shown that DNA CT, irrespective of the oxidant or reductant used to initiate the chemistry, can occur over long molecular distances but can be exquisitely sensitive to perturbations in the base pair stack. 

Here we review some of the critical characteristics of DNA charge transport chemistry, taking examples from a range of systems, and consider these characteristics in the context of their mechanistic implications. This review is not intended to be exhaustive but instead to be illustrative. For instance, we describe studies involving measurements in solution using pendant photooxidants to inject holes, conductivity studies with covalently modified assemblies, and electrochemical studies on DNA-modified electrodes. We do not focus in detail on the differences amongst these constructs but instead on their similarities. It is the similarity among these various systems that allows us to consider different mechanisms to describe DNA CT. Thus we review also the various mechanisms for DNA CT that have been put forth and attempt to reconcile these mechanistic proposals with the many disparate measurements of DNA CT. Certainly the debate among researchers has shifted from "is DNA CT possible?" to "how does it work?". This review intends to explore this latter question in detail.

Mechanisms for DNA Charge Transport

The dynamics of the electric fields in the interior of DNA are measured by using oligonucleotides in which a native base pair is replaced by a dye molecule (coumarin 102) whose emission spectrum is sensitive to the local electric field. Time-resolved measurements of the emission spectrum have been extended to a six decade time range (40 fs to 40 ns) by combining results from time-correlated photon counting, fluorescence up-conversion, and transient absorption. Recent results showed that when the reporter is placed in the center of the oligonucleotide, the dynamics are very broadly distributed over this entire time range and do not show specific time constants associated with individual processes (Andreatta, D.; et al. J. Am. Chem. Soc. 2005, 127, 7270). This paper examines an oligonucleotide with the reporter near its end. The broadly distributed relaxation seen before remains with little attenuation. In addition, a new relaxation with a well-defined relaxation time of 5 ps appears. This process is assign...

Ultrafast Dynamics in DNA: “Fraying” at the End of the Helix

Fluorescence correlation spectroscopy (FCS) is an ideal tool for measuring molecular diffusion and size under extremely dilute conditions. However, the power of FCS has not been utilized to its best to measure diffusion and size parameters of complex chemical systems. Here, we apply FCS to measure the size, and, most importantly, the size distribution and polydispersity of a supramolecular nanostructure (i.e., microemulsion droplets, MEDs) in dilute solution. It is shown how the refractive index mismatch of a solution can be corrected in FCS to obtain accurate size parameters of particles, bypassing the optical matching problem of light scattering techniques that are used often for particle-size measurements. We studied the MEDs of 13 different W0 values from 2 to 50 prepared in a ternary mixture of water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, with sulforhodamine-B as a fluorescent marker. We find that, near the optical matching point of MEDs, the dynamic light scattering (DLS) mea...

Fluorescence Correlation Spectroscopy: An Efficient Tool for Measuring Size, Size-Distribution and Polydispersity of Microemulsion Droplets in Solution

Time-resolved Stokes-shift experiments measure the dynamics of biomolecules and of the perturbed solvent near them on subnanosecond time scales, but molecular dynamics simulations are needed to provide a clear interpretation of the results. Here we show that simulations using standard methods quantitatively reproduce the main features of TRSS experiments in DNA and provide a molecular assignment for the dynamics. The simulations reproduce the magnitude and unusual power-law dynamics of the Stokes shift seen in recent experiments [Andreatta, D., et al. J. Am. Chem. Soc. 2005, 127, 7270]. A polarization model is introduced to eliminate cross-correlations between the different components contributing to the signal. Using this model, well-defined contributions of the DNA, water, and counterion to the experimental signal are extracted. Water is found to have the largest contribution and to be responsible for the power-law dynamics. The counterions have a smaller, but non-negligible, contribution with a time co...

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Dynamics of water and ions near DNA: comparison of simulation to time-resolved stokes-shift experiments.

The fluorescence anisotropy decay of two dyes merocyanine 540 and oxazine 1 has been studied in a polymer−surfactant aggregate containing poly(vinylpyrrolidone) (PVP) and sodium dodecyl sulfate (SDS). The results are analyzed in terms of the necklace model of the polymer−surfactant aggregate. The rotational motion of the probe is assumed to involve “wobbling-in-cone” along with translational motion along the micellar surface. It is observed that the presence of the polymer chains around the spherical SDS micelles causes significant retardation of both the wobbling motion as well as the translational motion of the two dyes. As a result, the wobbling and translational diffusion of the dyes in the PVP−SDS aggregate are slower than those in SDS micelle.

Sobhan Sen

Papers

Ultrafast Dynamics in DNA: “Fraying” at the End of the Helix

Fluorescence Correlation Spectroscopy: An Efficient Tool for Measuring Size, Size-Distribution and Polydispersity of Microemulsion Droplets in Solution

Dynamics of water and ions near DNA: comparison of simulation to time-resolved stokes-shift experiments.

Fluorescence Anisotropy Decay in Polymer-Surfactant Aggregates

Solvation dynamics of DCM in micelles