Nikita's+Article+Summaries

__April 22, 2009 Article Summary__

A group working at Arizona State University reported in 2005 a self-assembly of protein arrays which took place on the surface of a DNA nanostructure and was induced by aptamers. Single-stranded DNA molecules have the ability to self-assemble into branched motifs called tiles; these tiles have sticky ends that induce coagulation of the tiles and the formation of a large, multiple-tile DNA structure. This structure is a highly-ordered lattice and is customizable and programmable; the group induced the formation of protein arrays by attaching protein-specific aptamers to the DNA structure, to which the proteins would then readily and securely bind. To demonstrate this system, the group chose the well-developed aptamer sequence specific to thrombin. The group used one-dimensional triple-crossover DNA tiles; the tiles are similar to the ones developed below (March 13), with the exception of an added control DNA group on the opposite side of the aptamer group. A critical component in the chemistry of aptamer-protein binding, these two groups can be programmed to assemble with specific stem lengths, allowing for easier optimization. These tiles are on the nanomolecular scale and were analyzed by AFM imaging with a high degree of accuracy. After experimentation was completed, the group observed the tiles using the AFM and clearly discerned regular brighter spots on the tile. The distance from the tile and the dimensions of these spots were measured and were found to be in large agreement with the dimensions of thrombin; the distance between spots was the same as the spacing between aptamer groups, indicating a thrombin protein was bound to almost every aptamer group. Interestingly, the group noticed that the tiles would form parallel pairs with thrombin sandwiched in the middle; distance measurements of the separation between the pairs indicate that thrombin has a high potential to dimerize. The group also demonstrated that thrombin has two binding sites, both of which attach to the aptamer sequence, at two locations with certain triplets of oligonucleotides. The second binding site, that specific to heparin, was shown to form hydrogen bonds and ionic bonds with the aptamer sequence. The group concluded that aptamer-binding induces thrombin dimerization. Thus, based on the results of their experimentation, the group has demonstrated an effective and novel method to construct protein arrays by using DNA tiles with aptamers as binding-agents.

Liu, Yan, Chenxiang Lin, Hanying Li, Hao Yan. "Aptamer-Directed Self-Assembly of Protein Arrays on a DNA Nanostructure." Angewandte Chemie Internationale Edition (2005): 117, 4407-4412.

__April 8____, 2009 Article Summary__

A collaborative group from Brandeis University and the University of Texas at Austin reported in 2001 a new class of molecules termed aptamer beacons as a method of detecting the presence of ligands such proteins. While antibodies have previously been commonly used to detect protein targets, the immobilisation and adaptation of antibodies has proved to be difficult. This group then proceeded to demonstrate a replacement using aptamers. The group selected aptamer sequence specific for thrombin using relatively rapid //in vitro// selection: a specific oligonucleotide sequence is selected from a random pool of sequences, determined by the degree of binding for each individual sequence trial. To the 3’-end of this sequence the group attached the fluorophore fluorescein and to the 5’-end, they attached a fluorescence-quenching DABCYL-group. In the absence of target, the aptamer beacons form a stem-loop structure; at the end of the stem, the fluorescence-quencher is bound in close proximity with fluorophore. This structure exhibits little fluorescence; however when target binds to the loop target-binding site, the stem breaks apart and forms a more stable structure, and fluorescence is recovered. The group measured responses using circular dichroism spectrography over a thrombin concentration range of 0-120 nM; as controls, the experiment was repeated with coagulation factors IX and Xa, which are plasma serine proteases and are about 37% catalytically similar to thrombin. Results were positive, with fluorescence increasing 2.5-fold for the sequence with the shortest stem. The group performed subsequent experiments using MgCl2 and KCl to determine target-beacon interaction structures and found that metal ions have a profound effect on nucleic acid conformation and alter interactions; this can be alleviated by calibrating the signal in detection buffer prior to experimentation. The group also found that ssDNA-binding protein and lactate dehydrogenase non-specifically bind to the aptamer sequence and result in 15-fold fluorescence increases; this can be potentially mitigated by adding unlabeled, non-specific DNAs. The analysis of their results showed high association constants and consistent fluorescence shifts, indicating an effective detection method for thrombin.

Hamaguchi, Nobuko, Andrew Ellington, and Martin Stanton. “Aptamer Beacons for the Direct Detection of Proteins.” 15 Jun 2001. 8 Apr 2009 .

__March 19, 2009 Journal Summary__

Literature Review on “//Aptamer biosensor for protein detection using gold nanoparticles//”

A group working at the Peking University in Beijing, China, report a method of protein detection using protein-specific aptamers immobilized on gold nano-particles and complementary DNA strands tagged with a fluorescent dye. Originally used for their electronic, photonic, and catalytic properties, gold nano-particles of 5 and 10 nanometer sizes were used by this group as an effective immobilization site for aptamers; in fact, the group determined that on average 14 aptamers would bind to a single gold nano-particle. The aptamers, or single-stranded DNA which, in this case, would specifically bind to the fibrinogen-binding site (exosite I) of thrombin, were thiolated on the 3’ end; this SH-group then would covalently bind to the gold surface of the nano-particles. The aptamer included a 10-A-nucleotide-long spacer, followed by a sequence of six random nucleotides. To this random sequence of nucleotides, a complementary strand was developed that featured a fluorescent tag on the 5’ end. For this experiment, human alpha-thrombin was used as the target protein. Thrombin is not commercially sold labeled with a fluorescent tag, so the group devised a method to detect fluorescence upon thrombin binding. The labeled complementary strand was first hybridized to the immobilized aptamer, after which the solution of thrombin was flowed. The fluorescence of the complementary strand was quenched upon hybridization, and afterwards thrombin replaced this strand, thereby recovering the fluorescence. Fluorescence was measured using microscopic spectrometer under a controlled temperature. Fluorescence was successfully recovered, and the group proceeded to improve the method by determining a concentration of gold nanoparticles with immobilized aptamers that would best quench fluorescence and thus give a more distinct difference in amplitude upon thrombin binding. The group also ran the experiment using two other methods: one where an aptamer with a fluorescent tag would bind to thrombin and release the gold-nanoparticle with a non-labeled complementary strand of DNA, and the other where labeled adenosine would bind to thrombin. Of these three methods, the group determined that the immobilized aptamer worked best, although the hybridized aptamer (second method) worked well too. Adenosine-GNP interaction was determined to be very inhomogeneous and thus a poor detection limit; thus the third method of using adenosine was not effective. From mathematical fits to the data, a very high affinity and sensitive detection limit was determined for the best method, indicating an effective device for the detection of thrombin. Bibliography: Wang, Wenjuan et al. “Aptamer biosensor for protein detection using gold nanoparticles.” //Analytical Biochemistry// 373.2 (2008): 213-219. __March 13, 2009 Journal Summary__

Literature Review on //“Self-Assembled Signaling Aptamer DNA Arrays for Protein Detection”// A research group working at the Arizona State University has reported an effective new way of detecting the presence of thrombin through a self-assembled DNA-tile aptamer nanoarray. This DNA-tile array was assembled from single-stranded DNA oligonucleotides, which, when properly annealed together, self-assembled into DNA branched-junction building blocks, also known as DNA tiles. The self-assembly is facilitated by preferential self-hybridization of the “sticky” ends of each tile, thereby constructing a multi-tile DNA array on the nanomolecular level. Along with the DNA tile array, other single-stranded DNA oligonucleotides with subnanomolar affinities for proteins called aptamers were used. These aptamers were labeled with a florescent tag to the nucleotide adjacent to the protein-binding site. This method would then create multiple-fold increases of florescence upon binding to protein, specifically thrombin in this experiment. The florescence was then measured both quantitatively and qualitatively using confocal fluorescence microscope imaging. These aptamers were then linked to the DNA-tiles, and as the DNA-tiles self-assembled into a two-dimensional array, a high surface density of aptamers; the group reports that this large surface density allows for the detection of nanoscopic and even picoscopic detection of thrombin protein that is bound to the array. Fluorescence output was measured from an artificially attached 3-methylisoxanthopterin (3-MI), which is a fluorescent guanosine analogue with high sensitive to base stacking; this fluorescent tag was attached at position 7 on the aptamer where significant un-stacking upon protein binding has been shown to occur. The experiment was run with varying concentrations of thrombin protein (0 to 1200 nanomolar) and two different concentrations of DNA array (equivalent to 1 micromolar and 10 nanomolar thrombin-aptamer). With the larger concentration of DNA array, two-fold increase fluorescence intensity was observed; with the smaller concentration, a 60% increase was observed at a saturation concentration of 30 nanomolar thrombin. Dissociation constants were then calculated from a Langmuir fit; for this experiment, these constants were 2.5-fold larger than the current published effective dissociation constants for thrombin-binding aptamer, indicating that this system is a successful and effective in detecting thrombin protein. __Bibliographic Information:__ Lin, Chenxiang, Evaldas Katilius, Yan Liu, Junping Zhang, and Hao Yan. "Self-Assembled Signaling Aptamer DNA Arrays for Protein Detection." __Angewandte Chemie International Edition__ (2006): 5296-301.

__March 12, 2009 Journal Summary__

//Literature review on “////Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance” // March 12, 2009 A collaborative group from the Czech and Slovak Republics has demonstrated a sensitive device for detecting thrombin using thrombin-specific aptamers immobilized on a gold surface. An aptamer is linear sequence of single-stranded DNA or RNA, which under certain conditions tightly binds with proteins and even low-molecular weight compounds. This group used a predetermined sequence of DNA – 3’ GGTTGGTGTGGTTGG 5’ – that is known to bind specifically to thrombin, a large protein that is found in blood and is critical in blood coagulation; this aptamer was preassembled with three additional different spacers of random nucleotides that do not self-hybridize in order to lift the binding site above the immobilization surface and promote better binding. These aptamers were then immobilized on the gold surface, which already was coated in a layer of streptavidin, avidin, and neutravidin. The gold surface was part of a surface plamson resonance (SPR) sensor chip, through which a solution containing aptamers and later thrombin would be flowed. After the aptamers were immobilized onto each of the three surfaces, thrombin solution of varying concentrations was flowed through the SPR and the signal output recorded. As a control, a dendrimer- and a SH-gold-ended aptamers were used and the signal output recorded. From the recorded output, the group determined a nonlinear Langmuir fit and the subsequent constant of binding (KC). The group also determined sensitivity limits by using SPR as compared to other devices. The group determined high constants of binding for the three aptamer-thrombin binding experiments, indicating that the aptamer and thrombin are capable of binding under these conditions and that the SPR is an effective and sensitive way of measuring thrombin-aptamer binding. Ostatna, Veronika; Vaisocherova, Hana; Homola, Jiri; Hianik, Tibor. "Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance." __Anal Bioanal Chem__ (2008): 1861-869