Advances in aptamer evolution and engineering

2016 ◽  
Author(s):  
◽  
Khalid Kamal Alam
Keyword(s):  
High Throughput Sequencing ◽  
Target Selection ◽  
Three Dimensional ◽  
Rna Aptamers ◽  
Hiv Reverse Transcriptase ◽  
University Of Missouri ◽  
Selection Approach ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.

Aptamers against viral proteins as a tool to answer biological questions

2019 ◽  
Author(s):  
◽  
Carolina Camargo
Keyword(s):  
Lentiviral Vector ◽  
Rna Aptamers ◽  
Target Cells ◽  
University Of Missouri ◽  
Practical Applications ◽  
Intracellular Expression ◽  
The University ◽  
Further Development ◽  
Viral Surface

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] There are two experimental chapters in this dissertation in which the fundamental questions center around aptamers and viruses and how these two concepts interlace. The first experimental chapter (chapter two) seeks to utilize previously characterized aptamers against HIV-1 Reverse Transcriptase (RT) that will be delivered by a lentiviral vector and which intracellular expression from different human promoters was evaluated. The goal of this study was to identify important elements of vector design that will impact transgene expression in target cells. This study was based on the hypothesis that intracellular expression of RNA aptamers delivered by a lentiviral vector could offer a platform to enable adequate aptamer expression that would translate into viral suppression. And the second experimental chapter (chapter four) describes an in vitro 2'FY-RNA selection against Filoviral glycoproteins and outlines three different strategies that were followed to achieve selection of specific aptamers. Aptamers described in this chapter were able to recognize Ebolavirus glycoprotein ectodomain as well as in its native conformation displayed on the viral surface. Taking the observations obtained in this dissertation, aptamer technology could be expanded into further development for practical applications.

Investigating the role of arginine 21 in the structure and function of human [alpha]a-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh Shripad Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N- terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [subscript]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

Investigating the role of arginine 21 in the structure and function of human [alpha]A-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh S. Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N-terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [Alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [Alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [gamma]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

Sequence and secondary structural analysis of a novel class of RNA aptamers that inhibit reverse transcriptase

2012 ◽  
Author(s):  
◽  
Angela Whatley
Keyword(s):  
Reverse Transcriptase ◽  
Rna Aptamers ◽  
University Of Missouri ◽  
Hiv Rna ◽  
Cellular Assays ◽  
Subtype B ◽  
Single Target ◽  
Ic50 Values ◽  
The University ◽  
Major Target

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] RNA aptamers are synthetic RNAs selected for their ability to bind a molecular target. Reverse Transcriptase (RT) is the major target of antiretroviral therapy used in the treatment of HIV. RNA aptamers were selected to bind to RT of HIV-1 subtype B. Previous work showed aptamers from this selection inhibited various RT enzymatic activities, and have also been shown to inhibit viral replication in cellular assays. Most of the aptamers from this selection have predicted pseudoknot structures. The aptamers were classified into Pseudoknot families. Family 1 Pseudoknots (F1Pk) have a conserved UCCG sequence in stem one. Family 2 Pseudoknots (F2Pk) aptamers have CYGG (Y is pyrimidine either C or U). Aptamer secondary structural families are correlated with inhibitory behavior. F2Pk aptamers inhibit a broader range of RT subtypes compared to F1Pk. In the present work we present results of a screen into the inhibitory behavior of a novel family of anti RT aptamer from a previously well- studied pool. We found a group of aptamers that inhibit RT from subtype B very well and have novel secondary structure. These aptamers fold into stem loops with a highly conserved UCAA bulge. Aptamers in the UCAA family inhibit RT very well in primer extension assays and have IC50 values as low as 1.6nM. This work highlights the diversity of RNA secondary structures that are able to bind to a single target.

scholarly journals Renal reabsorption in 3D vascularized proximal tubule models

2019 ◽  
Vol 116 (12) ◽  
pp. 5399-5404 ◽  
Author(s):  
Neil Y. C. Lin ◽  
Kimberly A. Homan ◽  
Sanlin S. Robinson ◽  
David B. Kolesky ◽  
Nathan Duarte ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Disease Modeling ◽  
Three Dimensional ◽  
Kidney Tissue ◽  
Renal Reabsorption ◽  
Native Kidney ◽  
Glucose Reabsorption ◽  
Diseased State ◽  
And Function

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular–vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium–endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

scholarly journals Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

2020 ◽  
Vol 21 (22) ◽  
pp. 8774
Author(s):  
Natalia Komarova ◽  
Daria Barkova ◽  
Alexander Kuznetsov
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Combinatorial Libraries ◽  
Structure And Function ◽  
Huge Number ◽  
Systematic Evolution ◽  
And Function ◽  
Exponential Enrichment

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

scholarly journals An In Vitro Mixed Infection Model With Commensal and Pathogenic Staphylococci for the Exploration of Interspecific Interactions and Their Impacts on Skin Physiology

2021 ◽  
Vol 11 ◽  
Author(s):  
Katsunori Kohda ◽  
Xuan Li ◽  
Naoki Soga ◽  
Risa Nagura ◽  
Tie Duerna ◽  
...  
Keyword(s):  
Mixed Infection ◽  
Interspecific Interactions ◽  
Abiotic Factors ◽  
Three Dimensional ◽  
3D Culture ◽  
Infection Model ◽  
Protective Effects ◽  
Skin Physiology ◽  
And Function

The skin microbiota has been recognized to play an integral role in the physiology and pathology of the skin. The crosstalk between skin and the resident microbes has been extensively investigated using two-dimensional (2D) and three-dimensional (3D) cell cultures in vitro; however, skin colonization by multiple species and the effects of interspecific interactions on the structure and function of skin remains to be elucidated. This study reports the establishment of a mixed infection model, incorporating both commensal (Staphylococcus epidermidis) and pathogenic (Staphylococcus aureus) bacteria, based on a 3D human epidermal model. We observed that co-infecting the 3D epidermal model with S. aureus and S. epidermidis restricted the growth of S. aureus. In addition, S. aureus induced epidermal cytotoxicity, and the release of proinflammatory cytokines was attenuated by the S. aureus-S. epidermidis mixed infection model. S. epidermidis also inhibited the invasion of the deeper epidermis by S. aureus, eliciting protective effects on the integrity of the epidermal barrier. This 3D culture-based mixed infection model would be an effective replacement for existing animal models and 2D cell culture approaches for the evaluation of diverse biotic and abiotic factors involved in maintaining skin health.

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency

2020 ◽  
Vol 52 (03) ◽  
pp. 186-193 ◽  
Author(s):  
Fernanda Borchers Coeli-Lacchini ◽  
Livia M. Mermejo ◽  
Aline Faccioli Bodoni ◽  
Lucila Leico Kagohara Elias ◽  
Wilson Araújo Silva Jr ◽  
...  
Keyword(s):  
Three Dimensional ◽  
External Genitalia ◽  
Structural Modeling ◽  
Primary Amenorrhea ◽  
Compound Heterozygous ◽  
Hydroxylase Deficiency ◽  
Vitro Analysis ◽  
And Function

Abstract17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2–2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

Human endothelial cell morphology and autacoid expression

1995 ◽  
Vol 268 (4) ◽  
pp. H1613-H1620
Author(s):  
C. J. de Groot ◽  
V. A. Chao ◽  
J. M. Roberts ◽  
R. N. Taylor
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Endothelin 1 ◽  
And Function ◽  
Cellular Fibronectin ◽  
Cells Cultured

Human umbilical vein endothelial (HUVE) cells plated on plastic or gelatin-coated dishes grow as a “cobblestone” monolayer. By contrast, endothelial cells cultured on a complex matrix (e.g., Matrigel) form three-dimensional, capillary-like structures. In the current study, we verified the capillary phenotype of the latter structures and asked whether the morphological changes induced by extracellular matrix also affect human endothelial gene expression and function in vitro. Concentrations of cellular fibronectin, prostacyclin, and endothelin-1 were measured in the conditioned media by enzyme-linked immunosorbent and radioimmunoassays. Steady-state concentrations of HUVE mRNA were estimated by reverse transcription-polymerase chain reaction and quantified by Northern analyses to assess fibronectin and endothelin-1 gene expression. We found that the subjacent extracellular matrix affects the morphology, proliferation, and differentiation of HUVE cells in vitro. Cells cultured on gelatin were more mitotically active, expressed significantly less cellular fibronectin, made similar amounts of prostacyclin, and secreted significantly more endothelin-1 compared with the same cells grown on a Matrigel substrate.

scholarly journals Shared and Group-Specific Features of the Rotavirus RNA Polymerase Reveal Potential Determinants of Gene Reassortment Restriction

2009 ◽  
Vol 83 (12) ◽  
pp. 6135-6148 ◽  
Author(s):  
Sarah M. McDonald ◽  
Daniel Aguayo ◽  
Fernando D. Gonzalez-Nilo ◽  
John T. Patton
Keyword(s):  
Rna Polymerase ◽  
Protein A ◽  
Three Dimensional ◽  
Structural Data ◽  
Homology Model ◽  
Recognition Mechanism ◽  
Shell Protein ◽  
And Function ◽  
Gene Reassortment

ABSTRACT Rotaviruses (RVs) are nonenveloped, 11-segmented, double-stranded RNA viruses that are major pathogens associated with acute gastroenteritis. Group A, B, and C RVs have been isolated from humans; however, intergroup gene reassortment does not occur for reasons that remain unclear. This restriction might reflect the failure of the viral RNA-dependent RNA polymerase (RdRp; VP1) to recognize and replicate the RNA of a different group. To address this possibility, we contrasted the sequences, structures, and functions of RdRps belonging to RV groups A, B, and C (A-VP1, B-VP1, and C-VP1, respectively). We found that conserved amino acid residues are located within the hollow center of VP1 near the active site, whereas variable, group-specific residues are mostly surface exposed. By creating a three-dimensional homology model of C-VP1 with the A-VP1 crystallographic data, we provide evidence that these RV RdRps are nearly identical in their tertiary folds and that they have the same RNA template recognition mechanism that differs from that of B-VP1. Consistent with the structural data, recombinant A-VP1 and C-VP1 are capable of replicating one another's RNA templates in vitro. Nonetheless, the activity of both RdRps is strictly dependent upon the presence of cognate RV core shell protein A-VP2 or C-VP2, respectively. Together, the results of this study provide unprecedented insight into the structure and function of RV RdRps and support the notion that VP1 interactions may influence the emergence of reassortant viral strains.

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