2 research outputs found
A Force Sensor that Converts Fluorescence Signal into Force Measurement Utilizing Short Looped DNA
A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force
G‑Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription
G-Quadruplexes
(GQs) found within the promoter regions of genes
are known to mostly act as repressors of transcription. Here we report
a guanosine (G)-rich segment in the 3′-proximal promoter region
of human tyrosine hydroxylase (<i>TH</i>), which acts as
a necessary element for transcription. Tyrosine hydroxylase catalyzes
the rate-limiting step in the catecholamine biosynthesis and is linked
to several common neurological disorders such as Parkinson’s
and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the
human <i>TH</i> promoter contains multiple G-stretches that
are extremely well conserved among the primates but deviate in rodents,
which raises the possibility of variation in the GQ structures formed
in the two orders with the potential for a distinctive functional
outcome. Biochemical and biophysical studies, including single-molecule
Förster resonance energy transfer, indicate that the wtTH49
sequence can adopt multiple GQ structures by using different combinations
of G-stretches. A functional assay performed with 2.8 kb of the 3′-proximal
end of the <i>TH</i> promoter and a mutated version (TH49fm;
mutated wtTH49) that is unable to form any GQ structure indicates
that overall the GQ-enabling wtTH49 sequence is functionally necessary
and enhances human <i>TH</i> promoter activity by 5-fold
compared to that of the mutant. Two additional mutants, each of which
was designed to form distinct GQs, differentially affected reporter
gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49
GQ and lowers the level of reporter gene expression, although its
analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence
within the human <i>TH</i> promoter can form multiple GQ
structures, is a necessary element in transcription, and depending
on the utilized combination of G-stretches affects transcription in
different ways