15 research outputs found
RNA cleavage with formation of 2′,3′-cyclic phosphate and 5′-hydroxyl termini
<p><b>Copyright information:</b></p><p>Taken from " selection, characterization, and application of deoxyribozymes that cleave RNA"</p><p>Nucleic Acids Research 2005;33(19):6151-6163.</p><p>Published online 11 Nov 2005</p><p>PMCID:PMC1283523.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> This reaction can occur alone or with a catalyst such as a protein enzyme, ribozyme or deoxyribozyme. In most but not all cases, a divalent metal ion cofactor (M) is required to achieve an appreciable reaction rate
Equilibrium fluorescence titrations of P4–P6 derivatives labeled with pyrene at U107 and A114
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Fluorescence emission spectra (λ = 340 nm) showing representative data from 0 to 400 Mg (1× TB buffer, 35°C). The U107 A3–A7 and T6–T10 spectra were similar in shape to A5 (data not shown). The low-wavelength spectral feature for U107-A2 was observed with varying intensity at all other tested nucleotide positions (see Supplementary Data). For each of the five A114 tethers, the fluorescence spectra were qualitatively similar to those for U107 with the same tether (see Supplementary Data). () Fitted titration curves for the A and T series of tethers at U107 and A114. The relative fluorescence intensity was determined at λ in all cases. See Materials and Methods for details of curve fitting. See Supplementary Data for [Mg] values
Equilibrium fluorescence titrations of P4–P6 derivatives labeled with pyrene at U247, U249, U253, A246, A256 or C240
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> For each of the five tethers, the fluorescence spectra were qualitatively similar to those for U107 that are depicted in (data not shown). See Supplementary Data for [Mg] values
Synthesizing pyrene-labeled P4–P6 by derivatization-ligation (path 1) or by derivatization-annealing (path 2)
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () A 15-mer or 24-mer oligoribonucleotide incorporating a single 2′-amino or 2′-(2-aminoethoxy) nucleotide is derivatized with pyrene as in and purified by PAGE. () To complete path 1, the 15-nt product from the derivatization step is ligated to the remaining 145 nt of P4–P6 using a DNA splint and T4 DNA ligase; the product is purified by PAGE. () To complete path 2, the 24-nt product from the derivatization step is annealed to the remaining 136 nt of P4–P6 without covalent ligation
Experiments with wild-type (GAAA) or modified (GAAA) tetraloops to show that pyrene fluorescence reports on P4–P6 tertiary folding for the T8 tether at A246, U247, U249 and U253
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> See text for details
Control experiments with reconstituted P6 region to show that A246-T8 pyrene fluorescence reports on P4–P6 tertiary folding
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> See text for details
Nondenaturing (native) polyacrylamide gel electrophoresis of pyrene-labeled P4–P6 RNAs
<p><b>Copyright information:</b></p><p>Taken from "Fluorescence of covalently attached pyrene as a general RNA folding probe"</p><p>Nucleic Acids Research 2006;34(1):152-166.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326244.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The Mg concentration used throughout each gel is shown on the right side of each image. Wt and unf denote unmodified wild-type P4–P6 and the unfolded control mutant (). () Representative data for U107 and A114 derivatives. The lane labels A and T denote P4–P6 derivatives with 2′-amino or 2′-tethered amino groups but lacking pyrene. () Representative equilibrium titration curves derived from the gel data. From these curves, the energetic consequences of pyrene derivatization may be computed (). The ΔΔ°′ values ranged from ∼0.4 kcal/mol (U107-A5 and T8) to ∼3–4 kcal/mol (U247-T8). For additional titration curves and ΔΔ°′ values, see Supplementary Data. () Data for U247, U249 and U253 derivatives. The wt and unf samples additionally labeled ‘ann’ were prepared by annealing the 24-mer RNA oligonucleotide to the 136-nt remainder of P4–P6 as in . The arrow on the left indicates the band corresponding to the unannealed 136-nt RNA that is particularly prominent at low Mg concentrations (see Materials and Methods). () Data for A246 and A256 derivatives
Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides
Dehydroalanine
(Dha) is a nonproteinogenic electrophilic amino
acid that is a synthetic intermediate or product in the biosynthesis
of several bioactive cyclic peptides such as lantibiotics, thiopeptides,
and microcystins. Dha also enables labeling of proteins and synthesis
of post-translationally modified proteins and their analogues. However,
current chemical approaches to introducing Dha into peptides have
substantial limitations. Using in vitro selection, here we show that
DNA can catalyze Zn<sup>2+</sup> or Zn<sup>2+</sup>/Mn<sup>2+</sup>-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit
pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two
new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1
and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on
the model hexapeptide substrate that was used during selection. Using
DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer
peptide. Subsequent base-promoted intramolecular cyclization of homocysteine
into Dha formed a stable cystathionine (thioether) analogue of the
complement inhibitor compstatin. These findings establish the fundamental
catalytic ability of DNA to eliminate phosphate from pSer to form
Dha and suggest that with further development, pSer lyase deoxyribozymes
will have broad practical utility for site-specific enzymatic synthesis
of Dha from pSer in peptide substrates
DNA Catalysts with Tyrosine Kinase Activity
We
show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate
tyrosine residues of peptides. Using in vitro selection, we identified
deoxyribozymes that transfer the γ-phosphoryl group from a 5′-triphosphorylated
donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor
of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use
pppRNA were identified from each of N<sub>30</sub>, N<sub>40</sub>, and N<sub>50</sub> random-sequence pools. Each deoxyribozyme requires
Zn<sup>2+</sup>, and most additionally require Mn<sup>2+</sup>. The
deoxyribozymes have little or no selectivity for the amino acid identities
near the tyrosine, but they are highly selective for phosphorylating
tyrosine rather than serine. Analogous GTP-dependent DNA catalysts
were identified and found to have apparent <i>K</i><sub>m</sub>(GTP) as low as ∼20 μM. These findings establish
that DNA has the fundamental catalytic ability to phosphorylate the
tyrosine side chain of a peptide substrate
Selective Aptamers for Detection of Estradiol and Ethynylestradiol in Natural Waters
We used in vitro selection to identify
new DNA aptamers for two
endocrine-disrupting compounds often found in treated and natural
waters, 17β-estradiol (E2) and 17α-ethynylestradiol (EE).
We used equilibrium filtration to determine aptamer sensitivity/selectivity
and dimethyl sulfate (DMS) probing to explore aptamer binding sites.
The new E2 aptamers are at least 74-fold more sensitive for E2 than
is a previously reported DNA aptamer, with dissociation constants
(<i>K</i><sub>d</sub> values) of 0.6 μM. Similarly,
the EE aptamers are highly
sensitive for EE, with <i>K</i><sub>d</sub> of 0.5–1.0
μM. Selectivity values indicate that the E2 aptamers bind E2
and a structural analogue, estrone (E1), equally well and are up to
74-fold selective over EE. One EE aptamer is 53-fold more selective
for EE over E2 or E1, but the other binds EE, E2, and E1 with similar
affinity. The new aptamers do not lose sensitivity or selectivity
in natural water from a local lake, despite the presence of natural
organic matter (∼4 mg/L TOC). DMS probing suggests that E2
binding occurs in relatively flexible single-stranded DNA regions,
an important finding for rational redesign of aptamers and their incorporation
into sensing platforms. This is the first report of aptamers with
strong selectivity for E2 and E1 over EE, or with strong selectivity
for EE over E2 and E1. Such selectivity is important for achieving
the goal of creating practically useful DNA-based sensors that can
distinguish structurally similar estrogenic compounds in natural waters