Owing to its great threat to human health and environment, Pb2+pollution has been recognized as a major public problem by the World Health Organization (WHO). Many DNA aptamers have been utilized in the development of Pb2+-detection sensors, but the underlying mechanisms remain elusive. Here, we report three Pb2+-complexed structures of the thrombin binding aptamer (TBA). These high-resolution crystal structures showed that TBA forms intramolecular G-quadruplex and Pb2+is bound by the two G-tetrads in the center. Compared to K+-stabilized G-quadruplexes, the coordinating distance between Pb2+and the G-tetrads are much shorter. The T3T4 and T12T13 linkers play important roles in dimerization and crystallization of TBA, but they are changeable for Pb2+-binding. In combination with mutagenesis and CD spectra, the G8C mutant structure unraveled that the T7G8T9 linker of TBA is also variable. In addition to expansion of the Pb2+-binding aptamer sequences, our study also set up one great example for quick and rational development of other aptamers with similar or optimized binding activity.
Cadmium (Cd) is one of the most toxic heavy metals. Exposure to Cd can impair the functions of the kidney, respiratory system, reproductive system and skeletal system. Cd2+-binding aptamers have been extensively utilized in the development of Cd2+-detecting devices; however, the underlying mechanisms remain elusive. This study reports four Cd2+-bound DNA aptamer structures, representing the only Cd2+-specific aptamer structures available to date. In all the structures, the Cd2+-binding loop (CBL-loop) adopts a compact, double-twisted conformation and the Cd2+ ion is mainly coordinated with the G9, C12 and G16 nucleotides. Moreover, T11 and A15 within the CBL-loop form one regular Watson–Crick pair and stabilize the conformation of G9. The conformation of G16 is stabilized by the G8–C18 pair of the stem. By folding and/or stabilizing the CBL-loop, the other four nucleotides of the CBL-loop also play important roles in Cd2+ binding. Similarly to the native sequence, crystal structures, circular dichroism spectrum and isothermal titration calorimetry analysis confirm that several variants of the aptamer can recognize Cd2+. This study not only reveals the underlying basis for the binding of Cd2+ ions with the aptamer, but also extends the sequence for the construction of novel metal–DNA complex.
more » « less- Award ID(s):
- 1845486
- NSF-PAR ID:
- 10405238
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 9
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 4625-4636
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R
W ITVTI – loop – KKIRVW E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RW ITVTI‐(GGGGKK)2GGGG‐KKIRVW E) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RW ITVRIW ‐(GGGGKK)2GGGG‐W KTIRVW E) supports a remarkably stable β‐sheet (ΔG U = 6.3 kJ/mol at 295 K,T m = ∼55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR. -
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Abstract Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that
E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition. -
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This data set for the manuscript entitled "Design of Peptides that Fold and Self-Assemble on Graphite" includes all files needed to run and analyze the simulations described in the this manuscript in the molecular dynamics software NAMD, as well as the output of the simulations. The files are organized into directories corresponding to the figures of the main text and supporting information. They include molecular model structure files (NAMD psf or Amber prmtop format), force field parameter files (in CHARMM format), initial atomic coordinates (pdb format), NAMD configuration files, Colvars configuration files, NAMD log files, and NAMD output including restart files (in binary NAMD format) and trajectories in dcd format (downsampled to 10 ns per frame). Analysis is controlled by shell scripts (Bash-compatible) that call VMD Tcl scripts or python scripts. These scripts and their output are also included.
Version: 2.0
Changes versus version 1.0 are the addition of the free energy of folding, adsorption, and pairing calculations (Sim_Figure-7) and shifting of the figure numbers to accommodate this addition.
Conventions Used in These Files
===============================Structure Files
----------------
- graph_*.psf or sol_*.psf (original NAMD (XPLOR?) format psf file including atom details (type, charge, mass), as well as definitions of bonds, angles, dihedrals, and impropers for each dipeptide.)- graph_*.pdb or sol_*.pdb (initial coordinates before equilibration)
- repart_*.psf (same as the above psf files, but the masses of non-water hydrogen atoms have been repartitioned by VMD script repartitionMass.tcl)
- freeTop_*.pdb (same as the above pdb files, but the carbons of the lower graphene layer have been placed at a single z value and marked for restraints in NAMD)
- amber_*.prmtop (combined topology and parameter files for Amber force field simulations)
- repart_amber_*.prmtop (same as the above prmtop files, but the masses of non-water hydrogen atoms have been repartitioned by ParmEd)Force Field Parameters
----------------------
CHARMM format parameter files:
- par_all36m_prot.prm (CHARMM36m FF for proteins)
- par_all36_cgenff_no_nbfix.prm (CGenFF v4.4 for graphene) The NBFIX parameters are commented out since they are only needed for aromatic halogens and we use only the CG2R61 type for graphene.
- toppar_water_ions_prot_cgenff.str (CHARMM water and ions with NBFIX parameters needed for protein and CGenFF included and others commented out)Template NAMD Configuration Files
---------------------------------
These contain the most commonly used simulation parameters. They are called by the other NAMD configuration files (which are in the namd/ subdirectory):
- template_min.namd (minimization)
- template_eq.namd (NPT equilibration with lower graphene fixed)
- template_abf.namd (for adaptive biasing force)Minimization
-------------
- namd/min_*.0.namdEquilibration
-------------
- namd/eq_*.0.namdAdaptive biasing force calculations
-----------------------------------
- namd/eabfZRest7_graph_chp1404.0.namd
- namd/eabfZRest7_graph_chp1404.1.namd (continuation of eabfZRest7_graph_chp1404.0.namd)Log Files
---------
For each NAMD configuration file given in the last two sections, there is a log file with the same prefix, which gives the text output of NAMD. For instance, the output of namd/eabfZRest7_graph_chp1404.0.namd is eabfZRest7_graph_chp1404.0.log.Simulation Output
-----------------
The simulation output files (which match the names of the NAMD configuration files) are in the output/ directory. Files with the extensions .coor, .vel, and .xsc are coordinates in NAMD binary format, velocities in NAMD binary format, and extended system information (including cell size) in text format. Files with the extension .dcd give the trajectory of the atomic coorinates over time (and also include system cell information). Due to storage limitations, large DCD files have been omitted or replaced with new DCD files having the prefix stride50_ including only every 50 frames. The time between frames in these files is 50 * 50000 steps/frame * 4 fs/step = 10 ns. The system cell trajectory is also included for the NPT runs are output/eq_*.xst.Scripts
-------
Files with the .sh extension can be found throughout. These usually provide the highest level control for submission of simulations and analysis. Look to these as a guide to what is happening. If there are scripts with step1_*.sh and step2_*.sh, they are intended to be run in order, with step1_*.sh first.
CONTENTS
========The directory contents are as follows. The directories Sim_Figure-1 and Sim_Figure-8 include README.txt files that describe the files and naming conventions used throughout this data set.
Sim_Figure-1: Simulations of N-acetylated C-amidated amino acids (Ac-X-NHMe) at the graphite–water interface.
Sim_Figure-2: Simulations of different peptide designs (including acyclic, disulfide cyclized, and N-to-C cyclized) at the graphite–water interface.
Sim_Figure-3: MM-GBSA calculations of different peptide sequences for a folded conformation and 5 misfolded/unfolded conformations.
Sim_Figure-4: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 370 K.
Sim_Figure-5: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 295 K.
Sim_Figure-5_replica: Temperature replica exchange molecular dynamics simulations for the peptide cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) with 20 replicas for temperatures from 295 to 454 K.
Sim_Figure-6: Simulation of the peptide molecule cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) in free solution (no graphite).
Sim_Figure-7: Free energy calculations for folding, adsorption, and pairing for the peptide CHP1404 (sequence: cyc(GTGSGTG-GPGG-GCGTGTG-SGPG)). For folding, we calculate the PMF as function of RMSD by replica-exchange umbrella sampling (in the subdirectory Folding_CHP1404_Graphene/). We make the same calculation in solution, which required 3 seperate replica-exchange umbrella sampling calculations (in the subdirectory Folding_CHP1404_Solution/). Both PMF of RMSD calculations for the scrambled peptide are in Folding_scram1404/. For adsorption, calculation of the PMF for the orientational restraints and the calculation of the PMF along z (the distance between the graphene sheet and the center of mass of the peptide) are in Adsorption_CHP1404/ and Adsorption_scram1404/. The actual calculation of the free energy is done by a shell script ("doRestraintEnergyError.sh") in the 1_free_energy/ subsubdirectory. Processing of the PMFs must be done first in the 0_pmf/ subsubdirectory. Finally, files for free energy calculations of pair formation for CHP1404 are found in the Pair/ subdirectory.
Sim_Figure-8: Simulation of four peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) where the peptides are far above the graphene–water interface in the initial configuration.
Sim_Figure-9: Two replicates of a simulation of nine peptide molecules with the sequence cyc(GTGSGTG-GPGG-GCGTGTG-SGPG) at the graphite–water interface at 370 K.
Sim_Figure-9_scrambled: Two replicates of a simulation of nine peptide molecules with the control sequence cyc(GGTPTTGGGGGGSGGPSGTGGC) at the graphite–water interface at 370 K.
Sim_Figure-10: Adaptive biasing for calculation of the free energy of the folded peptide as a function of the angle between its long axis and the zigzag directions of the underlying graphene sheet.
This material is based upon work supported by the US National Science Foundation under grant no. DMR-1945589. A majority of the computing for this project was performed on the Beocat Research Cluster at Kansas State University, which is funded in part by NSF grants CHE-1726332, CNS-1006860, EPS-1006860, and EPS-0919443. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562, through allocation BIO200030. -
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Abstract Nucleic acid aptamers selected for thrombin binding have been previously shown to possess anticoagulant activity; however, problems with rapid renal clearance and short circulation half‐life have prevented translation to clinical usefulness. Here, a family of self‐folding, functional RNA origami molecules bearing multiple thrombin‐binding RNA aptamers and showing significantly improved anticoagulant activity is described. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures are designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'‐fluoro‐modified C‐ and U‐nucleotides is shown to increase nuclease resistance and stability during long‐term storage. Specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA is demonstrated. The RNA origami constructs show anticoagulant activity sevenfold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity is maintained after at least 3 months of storage in buffer at 4 °C. Additionally, inhibition of thrombin is shown to be reversed by addition of single‐stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.
