8 research outputs found
Exceptionally rapid oxime and hydrazone formation promoted by catalytic amine buffers with low toxicity
Hydrazone and oxime bond formation between Ī±-nucleophiles (e.g. hydrazines, alkoxy-amines) and carbonyl compounds (aldehydes and ketones) is convenient and is widely applied in multiple fields of research. While the reactants are simple, a substantial drawback is the relatively slow reaction at neutral pH. Here we describe a novel molecular strategy for accelerating these reactions, using bifunctional buffer compounds that not only control pH but also catalyze the reaction. The buffers can be employed at pH 5-9 (5-50 mM) and accelerate reactions by several orders of magnitude, yielding second-order rate constants of >10 M-1s-1. Effective bifunctional amines include 2-(aminomethyl)imidazoles and N,N-dimethylethylenediamine. Unlike previous diaminobenzene catalysts, the new buffer amines are found to have low toxicity to human cells, and can be used to promote reactions in cellular applications
Fingerprints of Modified RNA Bases from Deep Sequencing Profiles
Posttranscriptional
modifications of RNA bases are not only found
in many noncoding RNAs but have also recently been identified in coding
(messenger) RNAs as well. They require complex and laborious methods
to locate, and many still lack methods for localized detection. Here
we test the ability of next-generation sequencing (NGS) to detect
and distinguish between ten modified bases in synthetic RNAs. We compare
ultradeep sequencing patterns of modified bases, including miscoding,
insertions and deletions (indels), and truncations, to unmodified
bases in the same contexts. The data show widely varied responses
to modification, ranging from no response, to high levels of mutations,
insertions, deletions, and truncations. The patterns are distinct
for several of the modifications, and suggest the future use of ultradeep
sequencing as a fingerprinting strategy for locating and identifying
modifications in cellular RNAs
ATP-Linked Chimeric Nucleotide as a Specific Luminescence Reporter of Deoxyuridine Triphosphatase
Nucleotide
surveillance enzymes play important roles in human health, by monitoring
damaged monomers in the nucleotide pool and deactivating them before
they are incorporated into chromosomal DNA or disrupt nucleotide metabolism.
In particular, deamination of cytosine, leading to uracil in DNA and
in the nucleotide pool, can be deleterious, causing DNA damage. The
enzyme deoxyuridine triphosphatase (dUTPase) is currently under study
as a therapeutic and prognostic target for cancer. Measuring the activity
of this enzyme is important both in basic research and in clinical
applications involving this pathway, but current methods are nonselective,
detecting pyrophosphate, which is produced by many enzymes. Here we
describe the design and synthesis of a dUTPase enzyme-specific chimeric
dinucleotide (DUAL) that replaces the pyrophosphate leaving group
of the native substrate with ATP, enabling sensitive detection via
luciferase luminescence signaling. The DUAL probe functions sensitively
and selectively to quantify enzyme activities <i>in vitro</i> and in cell lysates. We further report the first measurements of
dUTPase activities in eight different cell lines, which are found
to vary by a factor of 7-fold. We expect that the new probe can be
of considerable utility in research involving this clinically significant
enzyme
Luminescent Carbon Dot Mimics Assembled on DNA
Nanometer-sized
fragments of carbon in the form of multilayer graphene
(ācarbon dotsā) have been under highly active study
for applications in imaging. While offering advantages of low toxicity
and photostability, such nanomaterials are inhomogeneous and have
limited wavelengths of emission. Here we address these issues by assembling
luminescent aromatic C16āC38 hydrocarbons together on a DNA
scaffold in homogeneous, soluble molecular compounds. Monomer deoxyribosides
of five different aromatic hydrocarbons were synthesized and assembled
into a library of 1296 different tetramer compounds on PEG-polystyrene
beads. These were screened for photostability and a range of emission
colors using 365 nm excitation, observing visible light (>400 nm)
emission. We identified a set of six oligomers (DNA-carbon assemblies,
DNA-CAs) with exceptional photostability that emit from 400 to 680
nm in water, with Stokes shifts of up to 110 nm, quantum yields ranging
from 0.01 to 0.29, and fluorescence lifetimes from 3 to 42 ns. In
addition, several of these DNA-CAs exhibited white emission in aqueous
solution. The molecules were used in multispectral cell imaging experiments
and were taken up into cells passively. The results expand the range
of emission properties that can be achieved in water with all-hydrocarbon
chromophores and establish the use of the DNA scaffold to arrange
carbon layers in homogeneous, rapidly synthesized assemblies
An Excimer Clamp for Measuring DamagedāBase Excision by the DNA Repair Enzyme NTH1
Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase-induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real-time detection of NTH1 activity inā
vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small-molecule inhibitor with sub-micromolar potency
Potent and Selective Inhibitors of 8-Oxoguanine DNA Glycosylase
The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 Ī¼M). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 Ī¼M). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 Ī¼M. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways