Ouyang W.,Syracuse University |
Ouyang W.,Yeshiva University |
Okaine S.,Syracuse University |
Okaine S.,Regeneron Pharmaceuticals Inc. |
And 5 more authors.
Biochemistry | Year: 2013
The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N., et al. (1998) Science 279, 384-388]. Thirteen residues with functional or structural significance were mutated individually to Ala (Asn5, Phe6, Val13, Phe16, Asn17, Gly19, Glu21, Ile24, Gln45, Met46, Gly22, Pro 31, and Gly40), and three salt bridge switch mutants exchanged Lys and Glu (Lys14-Glu21, Lys 33-Glu42, and Lys38-Glu51). Dissociation constants (Kd) determined by fluorescence titration and isothermal titration calorimetry were used to compare affinities of SL3 for the variant proteins to that for the wild type. The F16A (Phe16 to Ala) variant showed a 25-fold reduction in affinity, consistent with a loss of organized structure in f1, the protein's first zinc finger. I24A, Q45A, and M46A reduced affinity by 2-5-fold; these residues occupy nearly equivalent positions in f1 and f2. E21A increased affinity by 3-fold, perhaps because of the mutant's increased net positive charge. Among the salt bridge switch mutants, only K14E/E21K in f1 caused a substantial change in affinity (5-fold reduction), binding SL3 with a biphasic binding isotherm. Aside from these six variants, most of the mutations studied have relatively minor effects on the stability of the complex. We conclude that many side chain interactions in the wild-type complex contribute little to stability or can be compensated by new contacts in the mutants. © 2013 American Chemical Society.
Kupakuwana G.V.,Syracuse University |
Crill J.E.,AptaMatrix |
McPike M.P.,AptaMatrix |
Borer P.N.,Syracuse University |
PLoS ONE | Year: 2011
Background: Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. Methodology/Principal Findings: We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2-6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (K d = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000:1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (K d = 0.5 μM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. Conclusions/Significance: Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets. © 2011 Kupakuwana et al.
Mohammad M.M.,Syracuse University |
Iyer R.,Syracuse University |
Howard K.R.,Syracuse University |
McPike M.P.,AptaMatrix |
And 4 more authors.
Journal of the American Chemical Society | Year: 2012
One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize, or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration. © 2012 American Chemical Society.
Agency: Department of Defense | Branch: Air Force | Program: STTR | Phase: Phase I | Award Amount: 99.97K | Year: 2012
ABSTRACT: This phase I STTR project is a proof of concept study for developing near real-time, nanoparticle based sensors that detect the presence of specified toxins, pathogens, proteins, metabolites and small molecules. The specificity and sensitivity of this"nanosensor"is imparted by aptamer recognition that is engineered into a novel, patented technology we termed as an Alloswitch. These Alloswitch sensors will be embedded in nanoparticles to create a"nanosensor"that are capable of long term storage, easy to administer to patients, and facilitate entry and uptake by cells. BENEFIT: There is a clear need for detection of bio-terror agents and new options for treatment of affected military and civilian personnel. There are also military and civilian needs for rapid diagnostics in bodily fluids and cells. Our nanosensor platform will enable rapid detection of targeted microorganisms, viruses, proteins, and metabolites, and also provide therapeutic options. Detection can be made in bodily fluids or specifically chosen cell types. Success in these applications will be of tremendous commercial and societal value.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 176.78K | Year: 2016
DESCRIPTION provided by applicant Aptamers are nucleic acid based andquot probeandquot molecules that provide similar affinity and specificity as antibodies towards protein targets Aptamers have found utility in a wide variety of diagnostic and therapeutic applications In AptaMatrix In helped pioneer a new method for discovering aptamer probes called Acyclic Identification of Aptamer AIA that leveraged the sequencing capacity of Illumina high throughput sequencing platforms This Phase I SBIR project is focused on further improving the AIA method by integrating a post sequencing Illumina protein screening step secondary screen into the AIA workflow We have established three project aims to successfully complete this project within the Illumina flowcell test optimize a novel method for generating truncated single stranded DNA ssDNA clusters needed for optimal target protein binding on the sequencing system integrate and optimize established protocols and software needed to image DNA protein binding and validate the complete method by monitoring DNA protein binding affinities using a well characterised thrombin thrombin binding aptamer TBA DNA library system Completion of this project will establish a robust and highly valuable platform for rapid and highly efficient aptamer discovery and assessment In Phase II we plan to scale up this method on our GAiix platform to offer rapid aptamer discovery services and consider purchasing a next generation Illumina sequencing platform to implement protein imaging capabilities to accommodate higher sample throughput at a lower operational cost PUBLIC HEALTH RELEVANCE Aptamers are nucleic acid based andquot probeandquot molecules that provide similar affinity and specificity as antibodies towards protein targets This Phase I SBIR project is directed at improving AptaMatrixandapos s AIA aptamer discovery approach by integrating a post sequencing protein imaging on an Illumina sequencing platform