Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

Download Full-text

Interaction modulation through arrays of clustered methyl-arginine protein modifications

10.1101/289041 ◽

2018 ◽

Author(s):

Jonathan Woodsmith ◽

Victoria Casado-Medrano ◽

Nouhad Benlasfer ◽

Rebecca L. Eccles ◽

Saskia Hutten ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Structural Complexity ◽

Arginine Methylation ◽

Cellular Interactions ◽

Systematic Analysis ◽

Methyl Transferase ◽

Disordered Protein ◽

Interaction Interface ◽

Binding Mechanisms

AbstractSystematic analysis of human arginine methylation identifies two distinct signaling modes; either isolated modifications akin to canonical PTM regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA binding protein SYNCRIP were experimentally shown to function in concert providing a tunable protein interaction interface. Quantitative immuno-precipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyl-transferase PRMT1 was promoted by continual arginine stretches while interaction with the methyl-binding protein SMN1 was arginine content dependent irrespective of linear position within the unstructured region. This study highlights how highly-repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions.

Download Full-text

A general approach for stabilizing nanobodies for intracellular expression

10.1101/2021.04.06.438746 ◽

2021 ◽

Author(s):

John Dingus ◽

Jonathan C.Y. Tang ◽

Constance Cepko

Keyword(s):

Structural Complexity ◽

Great Majority ◽

Data Bank ◽

Live Cells ◽

Binding Function ◽

Alternative Modality ◽

Intracellular Expression ◽

Distinct Sequence ◽

Target Binding

AbstractConventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of originally intracellularly unstable nanobodies, without compromising target-binding function. Additionally, we identified distinct sequence features impacting nanobody intracellular stability, and demonstrated mutationally stabilized nanobody expression in vivo, in the murine retina and in E. coli. This work will allow for standardized nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.

Download Full-text

Conformation of eukaryotic ribosomal RNAs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076561 ◽

1983 ◽

Vol 41 ◽

pp. 592-593

Author(s):

M. Boublik ◽

G. Thornton ◽

G. Oostergetel ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

Molecular Weight ◽

Mass Measurement ◽

Carbon Film ◽

Structural Complexity ◽

Scanning Transmission Electron Microscope ◽

Electron Microscopic ◽

Pure Nitrogen ◽

Freeze Dried ◽

Scanning Transmission ◽

Partially Unfolded

Understanding the structural complexity of ribosomes and their role in protein synthesis requires knowledge of the conformation of their components - rRNAs and proteins. Application of dedicated scanning transmission electron microscope (STEM), electrical discharge of the support carbon film in an atmosphere of pure nitrogen, and determination of the molecular weight of individual rRNAs enabled us to obtain high resolution electron microscopic images of unstained freeze-dried rRNA molecules from BHK cells in a form suitable for evaluation of their 3-D structure. Preliminary values for the molecular weight of 28S RNA from the large and 18S RNA from the small ribosomal subunits as obtained by mass measurement were 1.84 x 106 and 0.97 x 106, respectively. Conformation of rRNAs consists, in general, of alternating segments of intramolecular hairpin stems and single stranded loops in a proportion which depends on their ionic environment, the Mg++ concentration in particular. Molecules of 28S RNA (Fig. 1) and 18S RNA (not shown) obtained by freeze-drying from a solution of 60 mM NH+4 acetate and 2 mM Mg++ acetate, pH 7, appear as partially unfolded coils with compact cores suggesting a high degree of ordered secondary structure.

Download Full-text

Observations of Frozen-Hydrated Microtubules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181270 ◽

1990 ◽

Vol 48 (1) ◽

pp. 504-505

Author(s):

D. Chrétien ◽

D. Job ◽

R.H. Wade

Keyword(s):

Fourier Transforms ◽

Structural Complexity ◽

Image Contrast ◽

Phase Behaviour ◽

Experimental Conditions ◽

Thin Sections ◽

Surface Lattice ◽

Cilia And Flagella ◽

Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

Download Full-text

Structural Aspects of Phonological Development

Language Speech and Hearing Services in Schools ◽

10.1044/0161-1461.1901.05 ◽

1988 ◽

Vol 19 (1) ◽

pp. 5-16 ◽

Cited By ~ 5

Author(s):

Karen E. Pollock ◽

Richard G. Schwartz

Keyword(s):

Clinical Management ◽

Structural Complexity ◽

Strong Preference ◽

Phonological Development ◽

Structural Position ◽

Phonological Disorders ◽

Syllabic Structure ◽

The Relationship ◽

Structural Aspects ◽

Target Words

The relationship between syllabic structure and segmental development was examined longitudinally in a child with a severe phonological disorder. Six speech samples were collected over a 4-year period (3:5 to 7:3). Analyses revealed gradual increases in the complexity and diversity of the syllable structures produced, and positional preferences for sounds within these forms. With a strong preference for [d] and [n] at the beginning of syllables, other consonants appeared first at the end of syllables. Implications for clinical management of phonological disorders include the need to consider both structural position and structural complexity in assessing segmental skills and in choosing target words for intervention.

Download Full-text

Anti-Diabetic Activity of a Mineraloid Isolate, in vitro and in Genetically Diabetic Mice

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000048 ◽

2011 ◽

Vol 81 (1) ◽

pp. 34-42 ◽

Cited By ~ 1

Author(s):

Joel Deneau ◽

Taufeeq Ahmed ◽

Roger Blotsky ◽

Krzysztof Bojanowski

Keyword(s):

Dna Microarrays ◽

Human Fibroblasts ◽

Diabetic Animal ◽

In Vitro Tests ◽

Diabetic Mice ◽

Fossil Material ◽

Expression Of Genes ◽

Enhanced Expression ◽

Genetically Diabetic Mice

Type II diabetes is a metabolic disease mediated through multiple molecular pathways. Here, we report anti-diabetic effect of a standardized isolate from a fossil material - a mineraloid leonardite - in in vitro tests and in genetically diabetic mice. The mineraloid isolate stimulated mitochondrial metabolism in human fibroblasts and this stimulation correlated with enhanced expression of genes coding for mitochondrial proteins such as ATP synthases and ribosomal protein precursors, as measured by DNA microarrays. In the diabetic animal model, consumption of the Totala isolate resulted in decreased weight gain, blood glucose, and glycated hemoglobin. To our best knowledge, this is the first description ever of a fossil material having anti-diabetic activity in pre-clinical models.

Download Full-text