skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Friday, March 22 until 6:00 AM ET on Saturday, March 23 due to maintenance. We apologize for the inconvenience.


Title: Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8‐oxo ‐7,8‐dihydropurine, and 8‐bromopurine analogues within A‐form duplexes of RNA
Abstract

Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8‐oxo‐7,8‐dihydropurine (common products of oxidation at the C8‐position of purines), and 8‐bromopurine (as probes to explore conformational changes), derivatives, namely 8‐oxoG, 8‐oxoI, 8‐oxoA, 8‐BrG, and 8‐BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid‐phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (Tm) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H‐bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti‐I:syn‐A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8‐oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble‐pair is only stabilized by G; and (d) 8‐oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

 
more » « less
Award ID(s):
1954639
NSF-PAR ID:
10202539
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biopolymers
Volume:
111
Issue:
12
ISSN:
0006-3525
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication. 
    more » « less
  2. Abstract

    8‐Oxoguanosine is the most common oxidatively generated base damage and pairs with complementary cytidine within duplex DNA. The 8‐oxoguanosine−cytidine lesion, if not recognized and removed, not only leads to G‐to‐T transversion mutations but renders the base pair being more vulnerable to the ionizing radiation and singlet oxygen (1O2) damage. Herein, reaction dynamics of a prototype Watson−Crick base pair [9MOG ⋅ 1MC]⋅+, consisting of 9‐methyl‐8‐oxoguanine radical cation (9MOG⋅+) and 1‐methylcystosine (1MC), was examined using mass spectrometry coupled with electrospray ionization. We first detected base‐pair dissociation in collisions with the Xe gas, which provided insight into intra‐base pair proton transfer of 9MOG⋅+ ⋅ 1MC[9MOG − HN1]⋅ ⋅ [1MC+HN3′]+and subsequent non‐statistical base‐pair separation. We then measured the reaction of [9MOG ⋅ 1MC]⋅+with1O2, revealing the two most probable pathways, C5‐O2addition and HN7‐abstraction at 9MOG. Reactions were entangled with the two forms of 9MOG radicals and base‐pair structures as well as multi‐configurations between open‐shell radicals and1O2(that has a mixed singlet/triplet character). These were disentangled by utilizing approximately spin‐projected density functional theory, coupled‐cluster theory and multi‐referential electronic structure modeling. The work delineated base‐pair structural context effects and determined relative reactivity toward1O2as [9MOG − H]⋅>9MOG⋅+>[9MOG − HN1]⋅ ⋅ [1MC+HN3′]+≥9MOG⋅+ ⋅ 1MC.

     
    more » « less
  3. 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 thatE. coliRNAP 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.

     
    more » « less
  4. Abstract

    Peptide nucleic acids (PNA) with extended isoorotamide containing nucleobases (Io) were designed for binding A–U base pairs in double‐stranded RNA. Isothermal titration calorimetry and UV thermal melting experiments revealed improved affinity for A–U using theIoscaffold in PNA. PNAs having four sequentialIoextended nucleobases maintained high binding affinity.

     
    more » « less
  5. RNA interactions are exceptionally strong and highly redundant. As such, nearly any two RNAs have the potential to interact with one another over relatively short stretches, especially at high RNA concentrations. This is especially true for pairs of RNAs that do not form strong self-structure. Such phenomena can drive liquid–liquid phase separation, either solely from RNA–RNA interactions in the presence of divalent or organic cations, or in concert with proteins. RNA interactions can drive multimerization of RNA strands via both base-pairing and tertiary interactions. In this article, we explore the tendency of RNA to form stable monomers, dimers, and higher order structures as a function of RNA length and sequence through a focus on the intrinsic thermodynamic, kinetic, and structural properties of RNA. The principles we discuss are independent of any specific type of biomolecular condensate, and thus widely applicable. We also speculate how external conditions experienced by living organisms can influence the formation of nonmembranous compartments, again focusing on the physical and structural properties of RNA. Plants, in particular, are subject to diverse abiotic stresses including extreme temperatures, drought, and salinity. These stresses and the cellular responses to them, including changes in the concentrations of small molecules such as polyamines, salts, and compatible solutes, have the potential to regulate condensate formation by melting or strengthening base-pairing. Reversible condensate formation, perhaps including regulation by circadian rhythms, could impact biological processes in plants, and other organisms. 
    more » « less