Fluorescence Labeling of Nucleic Acids

Wood, Sharla; Rueda, David

doi:10.1007/978-3-642-16712-6_507

Cited by 3 publications

(2 citation statements)

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“…An intermolecular GC base pair in the loop was flipped to prevent homodimer formation while maintaining the KC stability (Figure 1a and Supplementary Materials). RNAs were deprotected, purified and labeled as previously described ( 40 , 41 ). Briefly, deprotected RNAs were purified by denaturing gel electrophoresis (20% wt/vol polyacrylamide and 8 M urea) and diffusion elution against elution buffer (0.5 M NH 4 OAc and 0.1 mM EDTA) overnight at 4°C, followed by chloroform extraction, ethanol precipitation and C8 reverse-phase High Performance Liquid Chromatography (HPLC).…”

Section: Methodsmentioning

confidence: 99%

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

Mundigala

Feig

Ennifar

et al. 2014

Nucleic Acids Research

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The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg2+, but not by K+ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg2+ ions.

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Section: Methodsmentioning

confidence: 99%

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

Mundigala

Feig

Ennifar

et al. 2014

Nucleic Acids Research

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“…Many of the most common fluorophores are sub-kilodalton highly substituted aromatic hydrocarbons [ 6 , 7 ]. Importantly, organic fluorophores are also relatively easy to derivatize for enhanced biocompatibility and facile conjugation chemistries, thereby enabling site-specific labeling of various functional groups in proteins [ 8 – 10 ] and synthetically modified nucleic acids [ 11 – 13 ]. Some of the most common bioconjugation methods utilize maleimide or N-hydroxysuccinimide moieties to couple these fluorophores to thiols and primary amines within the biomolecule of interest via flexible aliphatic linkers to minimize the likelihood for impaired biochemical function [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

Sanders

Holmstrom

2021

Essays in Biochemistry

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The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole–dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).

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Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

et al. 2017

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The CRISPR–Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA–RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.

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Fluorescence Labeling of Nucleic Acids

Cited by 3 publications

References 0 publications

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

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