Site-Specific Labeling of DNA and RNA Using an Efficiently Replicated and Transcribed Class of Unnatural Base Pairs

Young Jun Seo; Denis A. Malyshev; Thomas Lavergne; Phillip Ordoukhanian; Floyd E. Romesberg

doi:10.1021/ja207907d

Targeting the ALS/FTD-associated A-DNA kink with anthracene-based metal complex causes DNA backbone straightening and groove contraction

Nucleic Acids Research ◽

10.1093/nar/gkab227 ◽

2021 ◽

Author(s):

Cyong-Ru Jhan ◽

Roshan Satange ◽

Shun-Ching Wang ◽

Jing-Yi Zeng ◽

Yih-Chern Horng ◽

...

Keyword(s):

Hot Spot ◽

Hydration Shell ◽

Repeat Motif ◽

Dna Duplex ◽

Base Pairs ◽

Dna And Rna ◽

Detailed Structural Analysis ◽

Small Molecule Compound ◽

Dna Backbone ◽

Locomotor Deficits

Abstract The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA–DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA–DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a ‘hot-spot’ for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.6109 ◽

1991 ◽

Vol 11 (12) ◽

pp. 6109-6115 ◽

Cited By ~ 14

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.

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Metal-mediated base pairs in parallel-stranded DNA

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.13.265 ◽

2017 ◽

Vol 13 ◽

pp. 2671-2681 ◽

Cited By ~ 22

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.

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Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA

Nucleic Acids Research ◽

10.1093/nar/23.19.3916 ◽

1995 ◽

Vol 23 (19) ◽

pp. 3916-3921 ◽

Cited By ~ 16

Author(s):

Parag V. Sahasrabudhe ◽

Richard T. Pon ◽

William H. Gmeiner

Keyword(s):

Duplex Dna ◽

Site Specific ◽

Dna And Rna

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

Nucleic Acids Research ◽

10.1093/nar/gni128 ◽

2005 ◽

Vol 33 (15) ◽

pp. e129-e129 ◽

Cited By ~ 49

Author(s):

K. Moriyama ◽

M. Kimoto ◽

T. Mitsui ◽

S. Yokoyama ◽

I. Hirao

Keyword(s):

Base Pairs ◽

Site Specific ◽

Rna Molecules

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The biofilm matrix scaffold of Pseudomonas species contains non-canonically base paired extracellular DNA and RNA

10.1101/527267 ◽

2019 ◽

Cited By ~ 2

Author(s):

Thomas Seviour ◽

Fernaldo Richtia Winnerdy ◽

Lan Li Wong ◽

Xiangyan Shi ◽

Sudarsan Mugunthan ◽

...

Keyword(s):

Viscoelastic Properties ◽

Extracellular Dna ◽

Chromosomal Dna ◽

Base Pairs ◽

Matrix Component ◽

Dna And Rna ◽

Pseudomonas Species ◽

The Matrix ◽

Nucleic Acid Conformation ◽

Biofilm Matrix

AbstractWhile extracellular DNA (eDNA) is recognized as a critical biofilm matrix component, it is not understood how it contributes to biofilm function. Here we isolate eDNA from Pseudomonas biofilms using ionic liquids, and discover that its key biophysical signatures, i.e. fluid viscoelasticity, nucleic acid conformation, and temperature and pH dependencies of gel to solution transitions, are maintained. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, revealed non-canonical Hoogsteen base pairs, triads or tetrads involving guanine and thymine or uracil. These were less abundant in chromosomal DNA and undetected as eDNA underwent gel-sol transition. Purine-rich RNA was present in the eDNA network, which potentially enables eDNA to be the main cross-linking exopolymer in the matrix through non-canonical nucleobase interactions. Our study suggests that Pseudomonas assemble extracellular DNA and RNA into a network with viscoelastic properties, which underpin their persistence and spreading, and may aid the development of more effective controls for biofilm-associated infections.

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The Smart Solution for DNA Removal in Biopharmaceutical Production by Benzonase Endonuclease

Journal of Applied Virology ◽

10.21092/jav.v2i1.26 ◽

2013 ◽

Vol 2 (1) ◽

Cited By ~ 1

Author(s):

Zhu Yang ◽

Mingxin Li ◽

Wen Chen ◽

Alexander Peters

Keyword(s):

Mammalian Cells ◽

Rna World ◽

Rapid Development ◽

World Health ◽

Base Pairs ◽

Biological Products ◽

Continuous Cell Lines ◽

Dna And Rna ◽

Biomedical Technologies ◽

Health Organization

<p>More and more mammalian cells especially continuous cell lines were used to produce vaccines and therapeutic biological products with the rapid development of biomedical technologies. Therefore, more attention should be paid from drug regulatory authorities to the safety of biological products especially quality control of residual DNA and RNA. World Health Organization has evaluated the methods commonly used in vaccine manufacture for their ability to reduce the biological activity of DNA. Benzonase endonuclease is frequently used during the production of vaccines and can degrade all nucleic acid sequences down to oligonucleotides of approximately 3 to 5 base pairs.</p>

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

10.1101/631432 ◽

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.

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