Figure 2 - uploaded by André Eichert
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Optimization of LNA crystal growth using different metal salts as an additive: (a) 10 mM MgCl 2 , (b) 10 mM CaCl 2 , (c) 10 mM CuCl 2 , (d) 10 mM ZnSO 4. The basic conditions in all setups consisted of 50 mM sodium cacodylate pH 6.0 and 1 M lithium sulfate. The crystals in (d) had approximate dimensions of 0.25 Â 0.25 Â 0.8 mm.
Source publication
Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as beta-D-2'-O,4'-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The beta-D-2'-O,4'-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the st...
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Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA a...
Detecting low-level clinically significant cancer-relevant somatic mutations can be difficult. Several technologies exist for detecting minority mutations. One method is locked nucleic acid (LNA) PCR. In this study, LNA probes were used to enhance the sensitivity for detecting FLT3 D835/I836 tyrosine kinase domain (TKD) mutations, the JAK2 V617F mu...
MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo....
Detection of single based genetic mutation by using oligonucleotide probes is one of the common methods of detecting single nucleotide polymorphisms at known loci. In this paper, we demonstrated a hybridization system which included a buffer solution that produced selective salt-induced effect and a locked nucleic acid modified 12 nt oligonucleotid...
Short single-stranded oligonucleotides called aptamers, often termed as chemical antibodies, have been developed as powerful alternatives to the traditional antibodies with respect to their obvious advantages like high specificity and affinity, longer shelf-life, easy manufacturing procedure, freedom to introduce chemical modifications for further...
Citations
... Nucleic acid aptamers are much smaller than antibodies. In recent years, there are many studies on the crystallization and X-ray diffraction analysis of aptamers or the complex of aptamers and enzyme [90][91][92][93]. It is an effective method to develop modified aptamers with higher affinity and selectivity according to the crystal structures. ...
Nucleic acid aptamers have minimal immunogenicity, high chemical synthesis production, low cost and high chemical stability when compared with antibodies. However, the susceptibility to nuclease degradation, rapid excretion through renal filtration and insufficient binding affinity hindered their development as drug candidates for therapeutic applications. In this review, we will discuss methods to conquer these challenges and highlight recent developments of chemical modifications and technological advances that may enable early aptamers to be translated into clinical therapeutics.
... Earlier, use of post-SELEX modification methods were mainly reported in studies on BNA aptamer development. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] However, in recent years, in vitro selection of BNA aptamers is preferred owing to the discovery of polymerases available for BNA-containing oligonucleotide syntheses and genetic engineering of these polymerases. ...
... Successful examples of 2',4'-BNA/LNA aptamers by post-SELEX modification have been reported. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Schmidt et al. developed an LNA aptamer, TTA1, specific for human tenascin-C (TN-C). 11 The original form TN-9 was selected from a 2'-fluoropyrimidine RNA library (Fig. 3A). ...
Recently, we achieved the first in vitro selection of 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA) aptamers. High-affinity thrombin-binding aptamers (TBAs) were obtained from DNA-based libraries containing 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the 5'-primer region, using the method of capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX). Furthermore, a similar selection protocol could provide TBAs that contain B/L nucleotides in both primer and random regions. We review technical challenges involved in the generation of various BNA libraries using analogs of B/L nucleoside-5'-triphosphate and polymerase variants and also discuss applications of these libraries to the selection of BNA (LNA) aptamers, as well as future prospects for their therapeutic and diagnostic uses.
... The short 7 bp LNA duplex, derived from the tRNASer microhelix for this study, exhibits a melting temperature of above 90°C, whereas the corresponding RNA has a Tm value of 45.0°C [9]. We have previously investigated the melting temperature of another LNA 7mer helix in comparison to its natural RNA counterpart [29]. Similarly, the LNA duplex possesses a Tm value of 84.3°C, whereas its corresponding RNA helix melts at 22.4°C. ...
“Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2
′
-
O
,4
′
-
C
-methylene-
β
-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery.
... As reported previously, we measured the melting curves of the RNA and LNA 7mer duplex to examine their thermostability [12]. Melting curves of the aptamers were recorded on a HP Agilent 8453 UV/visible diode array spectrophotometer (Agilent ChemStation software) in PBS using 2.5 lM aptamer. ...
... As has been reviewed, an increase of +2 to +10 °C can be observed per LNA building block added in strands hybridized to RNA [6]. As reported previously [12], we have investigated the melting temperature of the aptamer's 7mer RNA helix and compared the data to the LNA's 7mer counterpart. Whereas the RNA duplex possesses a T m value of 22 °C, the corresponding 'all LNA' duplex shows a T m value of 84 °C. ...
... Apart from antibodies, DNA-or RNA-aptamers have been reported to selectively bind ricin [261][262][263][264][265][266][267][268]. It has been proposed that they could be used as an alternative to antibody-based detection methods [269], but still their diagnostic value in protein detection, especially out of complex matrices, is limited. ...
Accidental and intended Ricinus communis intoxications in humans and animals have been known for centuries but the causative agent remained elusive until 1888 when Stillmark attributed the toxicity to the lectin ricin. Ricinus communis is grown worldwide on an industrial scale for the production of castor oil. As by-product in castor oil production ricin is mass produced above 1 million tons per year. On the basis of its availability, toxicity, ease of preparation and the current lack of medical countermeasures, ricin has gained attention as potential biological warfare agent. The seeds also contain the less toxic, but highly homologous Ricinus communis agglutinin and the alkaloid ricinine, and especially the latter can be used to track intoxications. After oil extraction and detoxification, the defatted press cake is used as organic fertilizer and as low-value feed. In this context there have been sporadic reports from different countries describing animal intoxications after uptake of obviously insufficiently detoxified fertilizer. Observations in Germany over several years, however, have led us to speculate that the detoxification process is not always performed thoroughly and controlled, calling for international regulations which clearly state a ricin threshold in fertilizer. In this review we summarize knowledge on intended and unintended poisoning with ricin or castor seeds both in humans and animals, with a particular emphasis on intoxications due to improperly detoxified castor bean meal and forensic analysis.
We studied the molecular details of DNA aptamer-ricin interactions. The toxic protein ricin molecules were immobilized on a Au(111) surface using a N-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in situ using the AFM tip modified with an antiricin aptamer. Computer simulation was used to illustrate the protein and aptamer structures, the single-molecule ricin images on a Au(111) surface, and the binding conformations of ricin-aptamer and ricin-antibody complexes. The various ricin conformations on a Au(111) surface were caused by the different lysine residues reacting with the NHS ester. It was also observed that most of the binding sites for aptamer and antibody on the A chains of ricin molecules were not interfered by the immobilization reaction. The different locations of the ricin binding sites to aptamer and antibody were also distinguished by AFM recognition images and interpreted by simulations.
