scholarly journals Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

Molecules ◽  
2012 ◽  
Vol 17 (3) ◽  
pp. 2855-2876 ◽  
Author(s):  
Nobuyuki Morohashi ◽  
Michiko Kimoto ◽  
Akira Sato ◽  
Rie Kawai ◽  
Ichiro Hirao
Keyword(s):  
Base Pair ◽  
Site Specific ◽  
Transcription System ◽  
Functional Components

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.

scholarly journals Site-specific incorporation of functional components into RNA by transcription using unnatural base pair systems

2009 ◽  
Vol 53 (1) ◽  
pp. 73-74 ◽  
Author(s):  
M. Kimoto ◽  
A. Sato ◽  
R. Kawai ◽  
S. Yokoyama ◽  
I. Hirao
Keyword(s):  
Base Pair ◽  
Site Specific ◽  
Functional Components

Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry

2012 ◽  
Vol 48 (88) ◽  
pp. 10835 ◽  
Author(s):  
Takumi Ishizuka ◽  
Michiko Kimoto ◽  
Akira Sato ◽  
Ichiro Hirao
Keyword(s):  
Click Chemistry ◽  
Base Pair ◽  
Site Specific ◽  
Rna Molecules ◽  
Transcription System

scholarly journals Supercoiling and looping promote DNA base accessibility and coordination among distant sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan M. Fogg ◽  
Allison K. Judge ◽  
Erik Stricker ◽  
Hilda L. Chan ◽  
Lynn Zechiedrich
Keyword(s):  
Mechanical Stress ◽  
Genomic Instability ◽  
Base Pair ◽  
Base Pairing ◽  
Complex Interplay ◽  
Site Specific ◽  
Torsional Strain ◽  
Dna Base ◽  
Dna Backbone ◽  
Negative Supercoiling

AbstractDNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity.

scholarly journals Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

1986 ◽  
Vol 6 (12) ◽  
pp. 4329-4334 ◽  
Author(s):  
P V Prasad ◽  
D Horensky ◽  
L J Young ◽  
M Jayaram
Keyword(s):  
Base Pair ◽  
Substrate Recognition ◽  
Binding Assays ◽  
Site Specific ◽  
Contact Points ◽  
Dyad Symmetry

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.

scholarly journals Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

2020 ◽  
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Base Pair ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

AbstractConjugation of RNAs with nanoparticles is of significant importance for its numerous applications in biology and medicine, which however remains challenging, especially for large ones. So far, the majority of RNA labeling rely on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nts. We here present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with gold nanoparticle (AuNP) empowered by expanded genetic alphabet transcription. We synthesize an amine-derivatized TPT3 (TPT3A), which are site-specifically incorporated into a 97-nt 3’SL RNA and a 719-nt mini genomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by standard in vitro transcription with expanded genetic alphabet containing the A-T, G-C natural base pairs and the TPT3-NaM unnatural base pair. TPT3 modification cause minimal structural perturbations to the RNAs by small angle X-ray scattering. The purified TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-NHS-Nanogold nanoparticles via the highly selective amine-NHS ester reaction and purified under non-denaturing conditions. We demonstrate the application of the AuNP-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The inter-nanoparticle distance distributions in the 3’SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus validate the applicability of this novel labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies, and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.Significance StatementWe present a site-specific labeling strategy for large RNAs by T7 transcription with expanded genetic alphabet containing TPT3-NaM unnatural base pair. The applicability of this labeling strategy is validated by X-ray scattering interferometry measurements on a 97-nt and a 719-nt RNAs. This strategy can be applicable to natural RNAs or artificial RNA nanostructures with sizes from tens up to thousands of nucleotides, or covalent conjugation of RNAs with other metal nanoparticles. The usage of a far upstream forward primer during PCR enables easy purification of RNA from DNA templates, the non-denaturing conditions for conjugation reactions and purification avoids potential large RNA misfolding. This labeling strategy expands our capability to site-specifically conjugate RNA with nanoparticles for many applications.

scholarly journals Human Milk Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation

2019 ◽  
Vol 149 (8) ◽  
pp. 1317-1325 ◽  
Author(s):  
Elisha Goonatilleke ◽  
Jincui Huang ◽  
Gege Xu ◽  
Lauren Wu ◽  
Jennifer T Smilowitz ◽  
...  
Keyword(s):  
Human Milk ◽  
Immunoglobulin A ◽  
Milk Proteins ◽  
Structure Stability ◽  
Mature Milk ◽  
Site Specific ◽  
Quantitative Changes ◽  
Protein Structure Stability ◽  
Analytical Tools ◽  
Functional Components

ABSTRACTBackgroundProteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown.ObjectiveThis study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation.DesignA tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified.Resultsα-Lactalbumin, β-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37 ± 1.33 g/L to 6.41 ± 0.72 g/L, 2.25 ± 0.86 g/L to 2.59 ± 0.78 g/L, 1.33 ± 0.44 g/L to 1.60 ± 0.39 g/L, and 0.09 ± 0.10 g/L to 0.11 ± 0.04 g/L, respectively; P < 0.002). α-Lactalbumin (37%), β-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased.ConclusionsProteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

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