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Results for “Oligonucleotide Probes”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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46 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS1997

CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast nuclear proteins. These results identify C/EBPdelta as a hormonally activated inducer of IGF-I gene transcription in osteoblasts and show that the HS3D element within IGF-I promoter 1 is a high affinity binding site for this protein.

NASA Discipline Musculoskeletal↗
NASA NTRS2003

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

Non-NASA Center↗
DOE OSTI2021

The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer

Aptamers are short, single-stranded DNA or RNA oligonucleotide molecules that function as synthetic analogs of antibodies and bind to a target molecule with high specificity. Aptamer affinity entirely depends on its tertiary structure and charge distribution. Therefore, length and structure optimization are essential for increasing aptamer specificity and affinity. Here, we present a general optimization procedure for finding the most populated atomistic structures of DNA aptamers. Based on the existed aptamer LC-18 for lung adenocarcinoma, a new truncated LC-18 (LC-18t) aptamer LC-18t was developed. A three-dimensional (3D) shape of LC-18t was reported based on small-angle X-ray scattering (SAXS) experiments and molecular modeling by fragment molecular orbital or molecular dynamic methods. Molecular simulations revealed an ensemble of possible aptamer conformations in solution that were in close agreement with measured SAXS data. The aptamer LC-18t had stronger binding to cancerous cells in lung tumor tissues and shared the binding site with the original larger aptamer. The suggested approach reveals 3D shapes of aptamers and helps in designing better affinity probes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Modification at the C2'-O-Position with 2-Methylbenzothiophene Induces Unique Structural Changes and Thermal Transitions on Duplexes of RNA and DNA

Oligonucleotides can be chemically modified for a variety of applications that include their use as biomaterials, in therapeutics, or as tools to understand biochemical processes, among others. This work focuses on the functionalization of oligonucleotides of RNA and DNA (12- or 14-nucleotides long) with methylbenzothiophene (BT), at the C2'-O-position, which led to unique structural features. Circular dichroism (CD) analyses showed that positioning the BT units on one strand led to significant thermal destabilization, while duplexes where each strand contained 4-BT rings formed a distinct arrangement with cooperativity/interactions among the modifications (evidenced from the appearance of a band with positive ellipticity at 235 nm). Interestingly, the structural arrays displayed increased duplex stabilization (>10 °C higher than the canonical analogue) as a function of [Na + ] with an unexpected structural rearrangement at temperatures above 50 °C. Density functional theory–polarizable continuum model (DFT-PCM) calculations were carried out, and the analyses were in agreement with induced structural changes as a function of salt content. A model was proposed where the hydrophobic surface allows for an internal nucleobase rearrangement into a more thermodynamically stable structure, before undergoing full denaturation, with increased heat. While this behavior is not common, B- to Z-form duplex transitions can occur and are dependent on parameters that were probed in this work, i.e., temperature, nature of modification, or ionic content. To take advantage of this phenomenon, we probed the ability of the modified duplexes to be recognized by Zα (an RNA binding protein that targets Z-form RNA) via electrophoretic analysis and CD. Interestingly, the protein did not bind to canonical duplexes of DNA or RNA; however, it recognized the modified duplexes, in a [monovalent/divalent salt] dependent manner. Overall, the findings describe methodology to attain unique structural motifs of modified duplexes of DNA or RNA, and control their behavior as a function of salt concentration. While their affinity to RNA binding proteins, and the corresponding mechanism of action, requires further exploration, the tunable properties can be of potential use to study this, and other, types of modifications. The novel arrays that formed, under the conditions described herein, provide a useful way to explore the structure and behavior of modified oligonucleotides, in general.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cooperative control of a DNA origami force sensor

Abstract Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A DNA molecular printer capable of programmable positioning and patterning in two dimensions

Nanoscale manipulation and patterning usually require costly and sensitive top-down techniques such as those used in scanning probe microscopies or in semiconductor lithography. DNA nanotechnology enables exploration of bottom-up fabrication and has previously been used to design self-assembling components capable of linear and rotary motion. In this work, we combine three independently controllable DNA origami linear actuators to create a nanoscale robotic printer. The two-axis positioning mechanism comprises a moveable gantry, running on parallel rails, threading a mobile sleeve. We show that the device is capable of reversibly positioning a write head over a canvas through the addition of signaling oligonucleotides. We demonstrate “write” functionality by using the head to catalyze a local DNA strand–exchange reaction, selectively modifying pixels on a canvas. Furthermore, this work demonstrates the power of DNA nanotechnology for creating nanoscale robotic components and could find application in surface manufacturing, biophysical studies, and templated chemistry.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2021

Carbon nanotube biocompatibility in plants is determined by their surface chemistry

Abstract Background Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Results Herein, we characterize the response of Arabidopsis thaliana to single walled carbon nanotube (SWNT) exposure with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that pristine SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this adverse reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Conclusions While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We use molecular markers to identify more biocompatible SWNT formulations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement. Graphical Abstract

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

NASA Discipline Plant Biology↗
NASA NTRS1995

PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world.

Cassell, Gail H.↗