Nucleic Acid (NA) nanotechnology is a rapidly emerging field demonstrating application of polynucleotides as a versatile biopolymer to fabricate nanostructures of various dimensions and shapes in a programmable and highly predictable way. The folding of DNA or RNA strands into a stable double helix configuration mainly relies on the Watson-Crick (Canonical) base pair composition (G=C and A-T or A-U in the case of RNA), base stacking, and metal ion concentrations. The thermodynamic parameters of DNA B-form helix formation and A-form helix of RNA can be computed using empirically defined sets of nearest neighboring parameters encompassed within the 2D structure predicting programs for example mfold, NUPAC. However, these programs are lacking parameters for a hybrid DNA/RNA base pairing and non-canonical base interactions. In this report, we focused our study to evaluate thermodynamic parameters of several in silico designed three-way junction (3WJ) DNA and hybrid DNA-RNA structural elements. The designed 3WJ motifs contain three helical stems linked with 4,3,2,1, and 0 single stranded Thymidine (T) or Uridine (U) nucleotides. We will report assembly efficiency of the 3WJs investigated by gel shift assay and thermodynamic parameters measured by UV-melting technique. Our experiments reveal that the amount of Ts and Us linkages in the three-way junction dictate the stability of the overall 3WJ conformations. This study is important as we expect it will contribute to the existing set of parameters used for NA structure prediction algorithms as well as provide a guidance for rational design of NA nanostructures.
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Prebiotic Origin of Pre‐RNA Building Blocks in a Urea “Warm Little Pond” Scenario
Urea appears to be a key intermediate of important prebiotic synthetic pathways. Concentrated pools of urea likely existed on the surface of the early Earth, as urea is synthesized in significant quantities from hydrogen cyanide or cyanamide (widely accepted prebiotic molecules), it has extremely high water solubility, and it can concentrate to form eutectics from aqueous solutions. We propose a model for the origin of a variety of canonical and non‐canonical nucleobases, including some known to form supramolecular assemblies that contain Watson‐Crick‐like base pairs.The dual nucleophilic‐electrophilic character of urea makes it an ideal precursor for the formation of nitrogenous heterocycles. We propose a model for the origin of a variety of canonical and noncanonical nucleobases, including some known to form supramolecular assemblies that contain Watson‐Crick‐like base pairs. These reactions involve urea condensation with other prebiotic molecules (e. g., malonic acid) that could be driven by environmental cycles (e. g., freezing/thawing, drying/wetting). The resulting heterocycle assemblies are compatible with the formation of nucleosides and, possibly, the chemical evolution of molecular precursors to RNA. We show that urea eutectics at moderate temperature represent a robust prebiotic source of nitrogenous heterocycles. The simplicity of these pathways, and their independence from specific or rare geological events, support the idea of urea being of fundamental importance to the prebiotic chemistry that gave rise to life on Earth.
more » « less- NSF-PAR ID:
- 10236168
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 21
- Issue:
- 24
- ISSN:
- 1439-4227
- Page Range / eLocation ID:
- p. 3504-3510
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization–enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.more » « less
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Abstract The cyanuric acid (CA) heterocycle forms supramolecular structures with adenine nucleobases/nucleosides and oligonucleotides, leading to speculation that they can act as forerunners to RNA. Herein, the assembly behavior of RNA containing CA and CA–ribose nucleoside was studied. Contrary to previous reports, CA in RNA and the CA‐ribonucleoside resulted in destabilization of supramolecular assemblies, which led to a reevaluation of the CA–adenine hexameric rosette structure. An unprecedented noncovalent supramolecular helicene structure is proposed to account for the striking difference in behavior, which has implications for novel paradigms for reorganizing the structures of nucleic acids, the synthesis of long helicenes, and pre‐RNA world paradigms. The results caution against extrapolating the self‐assembly behavior of individual heterocycles from the level of monomers to oligomers because the base‐paring properties of (non‐)canonical nucleobases are impacted by the type of oligomeric backbone to which they are attached.
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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.
