scholarly journals Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs

2005 ◽  
Vol 33 (15) ◽  
pp. e129-e129 ◽  
Author(s):  
K. Moriyama ◽  
M. Kimoto ◽  
T. Mitsui ◽  
S. Yokoyama ◽  
I. Hirao
Keyword(s):  
Base Pairs ◽  
Site Specific ◽  
Rna Molecules

scholarly journals PCR Amplification and Transcription for Site-Specific Labeling of Large RNA Molecules by a Two-Unnatural-Base-Pair System

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Michiko Kimoto ◽  
Rie Yamashige ◽  
Shigeyuki Yokoyama ◽  
Ichiro Hirao
Keyword(s):  
Base Pair ◽  
Pcr Amplification ◽  
Base Pairs ◽  
Dna Templates ◽  
Site Specific ◽  
Rna Molecules ◽  
Transcription System ◽  
Rna Transcripts ◽  
Pcr Method ◽  
Functional Components

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification andDsand pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modifiedPabases into RNA by T7 transcription. To prepareDs-containing DNA templates with long chains, theDs-Pxpair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containingDsat specific positions. Using theseDs-containing DNA templates and a biotin-linkedPasubstrate (Biotin-PaTP) as a modifiedPabase, 260-mer RNA transcripts containing Biotin-Paat a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining theDs-PxandDs-Papairs with modifiedPasubstrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.

Site-Specific Fluorescent Labeling of RNA Molecules by Specific Transcription Using Unnatural Base Pairs

2005 ◽  
Vol 127 (49) ◽  
pp. 17286-17295 ◽  
Author(s):  
Rie Kawai ◽  
Michiko Kimoto ◽  
Shuji Ikeda ◽  
Tsuneo Mitsui ◽  
Masayuki Endo ◽  
...  
Keyword(s):  
Fluorescent Labeling ◽  
Base Pairs ◽  
Site Specific ◽  
Rna Molecules

siRNA, miRNA, and shRNA: in vivo Applications

2008 ◽  
Vol 87 (11) ◽  
pp. 992-1003 ◽  
Author(s):  
P.N. Pushparaj ◽  
J.J. Aarthi ◽  
J. Manikandan ◽  
S.D. Kumar
Keyword(s):  
Metabolic Diseases ◽  
Small Interfering Rnas ◽  
Base Pairs ◽  
Rna Molecules ◽  
Rna Activation ◽  
Research Findings ◽  
Dental Diseases ◽  
Short Hairpin Rnas ◽  
Clinical Potential

RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects—such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety—need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ‘RNA activation’ (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.

scholarly journals Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115 ◽  
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

scholarly journals Metal-mediated base pairs in parallel-stranded DNA

2017 ◽  
Vol 13 ◽  
pp. 2671-2681 ◽  
Author(s):  
Jens Müller
Keyword(s):  
Nucleic Acid ◽  
Complex Formation ◽  
Transition Metal Ions ◽  
Dna Duplexes ◽  
Base Pairs ◽  
Experimental Conditions ◽  
Site Specific ◽  
Glycosidic Bonds ◽  
Double Helices ◽  
Significant Extension

In nucleic acid chemistry, metal-mediated base pairs represent a versatile method for the site-specific introduction of metal-based functionality. In metal-mediated base pairs, the hydrogen bonds between complementary nucleobases are replaced by coordinate bonds to one or two transition metal ions located in the helical core. In recent years, the concept of metal-mediated base pairing has found a significant extension by applying it to parallel-stranded DNA duplexes. The antiparallel-stranded orientation of the complementary strands as found in natural B-DNA double helices enforces a cisoid orientation of the glycosidic bonds. To enable the formation of metal-mediated base pairs preferring a transoid orientation of the glycosidic bonds, parallel-stranded duplexes have been investigated. In many cases, such as the well-established cytosine–Ag(I)–cytosine base pair, metal complex formation is more stabilizing in parallel-stranded DNA than in antiparallel-stranded DNA. This review presents an overview of all metal-mediated base pairs reported as yet in parallel-stranded DNA, compares them with their counterparts in regular DNA (where available), and explains the experimental conditions used to stabilize the respective parallel-stranded duplexes.

scholarly journals Altered Long Noncoding RNA Expression Profile in Multiple Myeloma Patients with Bisphosphonate-Induced Osteonecrosis of the Jaw

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Alessandro Allegra ◽  
Manuela Mania ◽  
Angela D’Ascola ◽  
Giacomo Oteri ◽  
Enrico Nastro Siniscalchi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Metabolic Pathways ◽  
Noncoding Rna ◽  
Protective Action ◽  
Noncoding Rnas ◽  
Osteonecrosis Of The Jaw ◽  
Base Pairs ◽  
Osteolytic Lesions ◽  
Ctrl Group ◽  
Rna Molecules

Bisphosphonates (BPs) are inhibitors of osteoclast-mediated bone resorption used for the treatment of multiple myeloma (MM) patients with osteolytic lesions. Bisphosphonate-induced osteonecrosis of the jaw (BONJ) is an infrequent drug-caused adverse event of these agents. Long noncoding RNAs (lncRNAs) are a set of more than 200 base pairs, noncoding RNA molecules, which are critical posttranscriptional regulators of gene expression. Our study was aimed at evaluating 17 lncRNAs, whose targets were previously validated as key elements in MM, bone metabolism, and angiogenesis in MM subjects without BONJ (MM group), in MM subjects with BONJ (BONJ group), and a group of healthy controls (CTRL group). Our results demonstrated a different lncRNA profile in BONJ patients compared to MM patients and controls. Two lncRNAs (DANCR and MALAT1) were both downregulated compared to controls and MM, twelve (HOTAIR, MEG3, TP73-AS1, HOTTIP, HIF1A-AS2, MANTIS, CTD-2201E18, CTD1-2003C8, R-471B22, RP1-43E13, RP11-553L6.5, and RP1-286D6) were overexpressed in MM with BONJ, and one (H19) was upregulated compared with only MM. Two lncRNAs (JHDMD1 and MTMR9LP) had higher expression, but these differences were not statistically significant. The examined lncRNAs target several genes and metabolic pathways. An altered lncRNA signature could contribute to the onset of BONJ or have a protective action. Targeting these lncRNAs could offer a possibility for the prevention or therapy of BONJ.

scholarly journals Site-Specific Chemical Labeling of Long RNA Molecules

2011 ◽  
Vol 22 (1) ◽  
pp. 95-100 ◽  
Author(s):  
Kasper Jahn ◽  
Eva Maria Olsen ◽  
Morten Muhlig Nielsen ◽  
Thomas Tørring ◽  
Reza MohammadZadegan ◽  
...  
Keyword(s):  
Chemical Labeling ◽  
Specific Chemical ◽  
Site Specific ◽  
Rna Molecules

scholarly journals Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

2019 ◽  
Author(s):  
Junetha Syed ◽  
Saravanan Palani ◽  
Scott T. Clarke ◽  
Zainab Asad ◽  
Andrew R. Bottrill ◽  
...  
Keyword(s):  
Genetic Code ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Glutathione S Transferase ◽  
Base Pairs ◽  
Site Specific ◽  
Green Fluorescent ◽  
Natural Amino Acids ◽  
Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

scholarly journals Interaction of influenza virus polymerase with viral RNA in the ‘corkscrew’ conformation

1999 ◽  
Vol 80 (10) ◽  
pp. 2565-2572 ◽  
Author(s):  
Ramon Flick ◽  
Gerd Hobom
Keyword(s):  
Influenza Virus ◽  
Base Pair ◽  
Short Range ◽  
Nucleotide Substitutions ◽  
Base Pairs ◽  
Stem Loop ◽  
Rna Molecules ◽  
Virus Rna ◽  
Core Elements ◽  
Complete Set

The influenza virus RNA (vRNA) promoter structure is known to consist of the 5′- and 3′-terminal sequences of the RNA, within very narrow boundaries of 16 and 15 nucleotides, respectively. A complete set of single nucleotide substitutions led to the previously proposed model of a binary hooked or ‘corkscrew’ conformation for the vRNA promoter when it interacts with the viral polymerase. This functional structure is confirmed here with a complete set of complementary double substitutions, of both the regular A:U and G:C type and also the G:U type of base-pair exchanges. The proposed structure consists of a six base-pair RNA rod in the distal element in conjunction with two stem–loop structures of two short-range base- pairs (positions 2–9; 3–8). These support an exposed tetranucleotide loop within each branch of the proximal element, in an overall oblique organization due to a central unpaired A residue at position 10 in the 5′ sequence. Long-range base-pairing between the entire 5′ and 3′ branches, as required for an unmodified ‘panhandle’ model, has been excluded for the proximal element, while it is known to represent the mode of interaction within the distal element. A large number of short-range base-pair exchanges in the proximal element constitute promoter-up mutations, which show activities several times above that of the wild- type in reporter gene assays. The unique overall conformation and rather few invariant nucleotides appear to be the core elements in vRNA recognition by polymerase and also in viral ribonucleoprotein packaging, to allow discrimination against the background of other RNA molecules in the cell.

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