Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

We describe a novel 3′-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2′-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I–dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3′-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies

A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella

A novel, site-specific, DNA backbone S-modification (phosphorothioation) has been discovered, but its in vivo function(s) have remained obscure. Here, we report that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from Escherichia coli, while protecting its own DNA by site-specific phosphorothioation. A cloned 15-kb gene cluster from S. enterica conferred both host-specific restriction and DNA S-modification on E. coli. Mutational analysis of the gene cluster proved unambiguously that the S-modification prevented host-specific restriction specified by the same gene cluster. Restriction activity required three genes in addition to at least four contiguous genes necessary for DNA S-modification. This functional overlap ensures that restriction of heterologous DNA occurs only when the host DNA is protected by phosphorothioation. Meanwhile, this novel type of host-specific restriction and modification system was identified in many diverse bacteria. As in the case of methylation-specific restriction systems, targeted inactivation of this gene cluster should facilitate genetic manipulation of these bacteria, as we demonstrate in Salmonella

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

© 2007 The Author et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in Nucleic Acids Research 35 (2007): 2107-2115, doi:10.1093/nar/gkm049.Trypanosomatids contain an unusual DNA base J (ß-D-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe2+ and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe2+ and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.This work was funded by a grant from the Netherlands Organization for Scientific Research and Chemical Sciences (NWO-CW) to P.B., NIH grant A1063523 to R.S. and NIH grant GM063584 to R.P.H

Nanostructured luminescently labeled nucleic acids

Important and emerging trends at the interface of luminescence, nucleic acids and nanotechnology are: (i) the conventional luminescence labeling of nucleic acid nanostructures (e.g. DNA tetrahedron); (ii) the labeling of bulk nucleic acids (e.g. single‐stranded DNA, double‐stranded DNA) with nanostructured luminescent labels (e.g. copper nanoclusters); and (iii) the labeling of nucleic acid nanostructures (e.g. origami DNA) with nanostructured luminescent labels (e.g. silver nanoclusters). This review surveys recent advances in these three different approaches to the generation of nanostructured luminescently labeled nucleic acids, and includes both direct and indirect labeling methods

A Histone Methyltransferase Modulates Antigenic Variation in African Trypanosomes

To evade the host immune system, several pathogens periodically change their cell-surface epitopes. In the African trypanosomes, antigenic variation is achieved by tightly regulating the expression of a multigene family encoding a large repertoire of variant surface glycoproteins (VSGs). Immune evasion relies on two important features: exposing a single type of VSG at the cell surface and periodically and very rapidly switching the expressed VSG. Transcriptional switching between resident telomeric VSG genes does not involve DNA rearrangements, and regulation is probably epigenetic. The histone methyltransferase DOT1B is a nonessential protein that trimethylates lysine 76 of histone H3 in Trypanosoma brucei. Here we report that transcriptionally silent telomeric VSGs become partially derepressed when DOT1B is deleted, whereas nontelomeric loci are unaffected. DOT1B also is involved in the kinetics of VSG switching: in ΔDOT1B cells, the transcriptional switch is so slow that cells expressing two VSGs persist for several weeks, indicating that monoallelic transcription is compromised. We conclude that DOT1B is required to maintain strict VSG silencing and to ensure rapid transcriptional VSG switching, demonstrating that epigenetics plays an important role in regulating antigenic variation in T. brucei

Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity