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DOE OSTI2017

HIV persistence in tissue macrophages of humanized myeloid-only mice during antiretroviral therapy

Despite years of fully suppressive antiretroviral therapy (ART), HIV persists in its hosts and is never eradicated. One major barrier to eradication is that the virus infects multiple cell types that may individually contribute to HIV persistence. Tissue macrophages are critical contributors to HIV pathogenesis; however, their specific role in HIV persistence during long-term suppressive ART has not been established. Using humanized myeloid-only mice (MoM), we demonstrate that HIV infection of tissue macrophages is rapidly suppressed by ART, as reflected by a rapid drop in plasma viral load and a dramatic decrease in the levels of cell-associated viral RNA and DNA. No viral rebound was observed in the plasma of 67% of the ART-treated animals at 7 weeks after ART interruption, and no replication-competent virus was rescued from the tissue macrophages obtained from these animals. In contrast, in a subset of animals (~33%), a delayed viral rebound was observed that is consistent with the establishment of persistent infection in tissue macrophages. Furthermore, these observations represent the first direct evidence, to our knowledge, of HIV persistence in tissue macrophages in vivo .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Exception to the Rule: Genomic Characterization of Naturally Occurring Unusual Vibrio cholerae Strains with a Single Chromosome

Here, the genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae , with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae . Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam , mutH , and mutL ; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae , Bunyaviridae , and Filoviridae Families

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5;-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1–/– and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1–/– and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this paper, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro. Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.

Biochemistry & Molecular Biology↗

DOE OSTI2021

Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Here, our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Highly Symmetric, Self-Assembling 3D DNA Crystals with Cubic and Trigonal Lattices

The rational design of nanoscopic DNA tiles has yielded highly ordered crystalline matter in 2D and 3D. The most well-studied 3D tile is the DNA tensegrity triangle, which is known to self-assemble into macroscopic crystals. However, contemporary rational design parameters for 3D DNA crystals nearly universally invoke integer numbers of DNA helical turns and Watson–Crick (WC) base pairs. Here, in this study, 24-bp edges are substituted into a previously 21-bp (two helical turns of DNA) tensegrity triangle motif to explore whether such unconventional motif can self-assemble into 3D crystals. The use of noncanonical base pairs in the sticky ends results in a cubic arrangement of tensegrity triangles with exceedingly high symmetry, assembling a lattice from winding helical axes and diamond-like tessellation patterns. Reverting this motif to sticky ends with Watson–Crick pairs results in a trigonal hexagonal arrangement, replicating this diamond arrangement in a hexagonal context. These results showcase that the authors can generate unexpected, highly complex, pathways for materials design by testing modifications to 3D tiles without prior knowledge of the ensuing symmetry. This study expands the rational design toolbox for DNA nanotechnology; and it further illustrates the existence of yet-unexplored arrangements of crystalline soft matter.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2013

The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2017

Development of Solid-State Nanopore Technology for Life Detection

Biomarkers for life on Earth are an important starting point to guide the search for life elsewhere. However, the search for life beyond Earth should incorporate technologies capable of recognizing an array of potential biomarkers beyond what we see on Earth, in order to minimize the risk of false negatives from life detection missions. With this in mind, charged linear polymers may be a universal signature for life, due to their ability to store information while also inherently reducing the tendency of complex tertiary structure formation that significantly inhibit replication. Thus, these molecules are attractive targets for biosignature detection as potential "self-sustaining chemical signatures." Examples of charged linear polymers, or polyelectrolytes, include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as synthetic polyelectrolytes that could potentially support life, including threose nucleic acid (TNA) and other xenonucleic acids (XNAs). Nanopore analysis is a novel technology that has been developed for singlemolecule sequencing with exquisite single nucleotide resolution which is also well-suited for analysis of polyelectrolyte molecules. Nanopore analysis has the ability to detect repeating sequences of electrical charges in organic linear polymers, and it is not molecule- specific (i.e. it is not restricted to only DNA or RNA). In this sense, it is a better life detection technique than approaches that are based on specific molecules, such as the polymerase chain reaction (PCR), which requires that the molecule being detected be composed of DNA.

Bywaters, K. B.↗

DOE OSTI2016

Final Report DE-SC0006634. Quantifying phenotypic and genetic diversity of Miscanthus sinensis as a resource for knowledge-based improvement of M. ×giganteus (M. sinensis × M. sacchariflorus)

Miscanthus is especially attractive as a bioenergy crop for temperate environments because it produces high yields, needs few inputs, and grows well during the cool weather of early spring and late fall when few warm-season grasses can. However, Miscanthus feedstock production for the emerging U.S. bioenergy industry and for existing demand in Europe is based on a single sterile, vegetatively propagated variety of M. ×giganteus. M. ×giganteus is an interspecific hybrid of the parental species M. sinensis and M. sacchariflorus. Prior to the current study, little information existed about the genetic diversity and breeding potential of either M. ×giganteus parental species. In the current project, we studied more than 600 accessions of M. sinensis from throughout its native range in China, Japan, and Korea, in addition to ornamental cultivars and U.S. naturalized populations. Using thousands of DNA markers, we identified seven geographically distinct genetic groups of M. sinensis. Notably, we found that the ornamental cultivars and U.S. naturalized populations were derived from only a subset of the Southern Japan group, indicating that our study greatly increased the genetic diversity available for breeding new biomass cultivars. Additionally, this new understanding of M. sinensis population structure could be used to predict which crosses may produce progeny with the greatest hybrid vigor. Replicated field trials were also established at multiple locations in North America and Asia. Data on traits of importance for biomass productivity, such as flowering time, yield and height, were taken. Analyses of the phenotypic data from the field trials along with the DNA markers allowed us to identify many marker-trait associations. These results will enable marker-assisted breeding, which will allow selection at the seedling stage rather than waiting two to three years to obtain phenotypic data. Thus, this study is expected to greatly increase the efficiency of breeding Miscanthus for improved adaptation and biomass yield.

09 BIOMASS FUELS↗

DOE OSTI2016

Thiosulfate oxidation by Thiomicrospira thermophila : Metabolic flexibility in response to ambient geochemistry

Previous studies of the stoichiometry of thiosulfate oxidation by colorless sulfur bacteria have failed to demonstrate mass balance of sulfur, indicating that unidentified oxidized products must be present. Here the reaction stoichiometry and kinetics under variable pH conditions during the growth of Thiomicrospira thermophila strain EPR85, isolated from diffuse hydrothermal fluids at the East Pacific Rise, is presented. At pH 8.0, thiosulfate was stoichiometrically converted to sulfate. At lower pH, the products of thiosulfate oxidation were extracellular elemental sulfur and sulfate. Here, we were able to replicate previous experiments and identify the missing sulfur as tetrathionate, consistent with previous reports of the activity of thiosulfate dehydrogenase. Tetrathionate was formed under slightly acidic conditions. Genomic DNA from T. thermophila strain EPR85 contains genes homologous to those in the Sox pathway ( soxAXYZBCDL ), as well as rhodanese and thiosulfate dehydrogenase. No other sulfur oxidizing bacteria containing sox(CD) 2 genes have been reported to produce extracellular elemental sulfur. If the apparent modified Sox pathway we observed in T. thermophila is present in marine Thiobacillus and Thiomicrospira species, production of extracellular elemental sulfur may be biogeochemically important in marine sulfur cycling.

58 GEOSCIENCES↗

DOE OSTI2014

RNA Catalysis, Thermodynamics and the Origin of Life

The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5'-polyphosphates (or their analogues) as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5'-triphosphates (or other analogously activated phosphodiester species) is inherently difficult. However, nucleotide 2',3'-cyclic phosphates are also phosphodiesters, and are the natural and abundant products of RNA degradation. These have previously been dismissed as viable substrates for prebiotic RNA synthesis. We propose that the arguments for their dismissal are based on a flawed assumption, and that nucleotide 2',3'-cyclic phosphates in fact possess several significant, advantageous properties that indeed make them particularly viable substrates for prebiotic RNA synthesis. An RNA World hypothesis based upon the polymerization of nucleotide 2',3'-cyclic phosphates possesses additional explanatory power in that it accounts for the observed ribozyme “fossil record”, suggests a viable mechanism for substrate transport across lipid vesicle boundaries of primordial proto-cells, circumvents the problems of substrate scarcity and implausible synthetic pathways, provides for a primitive but effective RNA replicase editing mechanism, and definitively explains why RNA, rather than DNA, must have been the original catalyst. Finally, our analysis compels us to propose that a fundamental and universal property that drives the evolution of living systems, as well as pre-biotic replicating molecules (be they composed of RNA or protein), is that they exploit chemical reactions that already possess competing kinetically-preferred and thermodynamically-preferred pathways in a manner that optimizes the balance between the two types of pathways.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Structural basis for a highly conserved RNA-mediated enteroviral genome replication

Abstract Enteroviruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3′ spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3′ spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Biochemistry & Molecular Biology↗

DOE OSTI2016

Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses

The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. Furthermore, a greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Evaluation of the Deletion of MGF110-5L-6L on Swine Virulence from the Pandemic Strain of African Swine Fever Virus and Use as a DIVA Marker in Vaccine Candidate ASFV-G-ΔI177L

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-DMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-DI177L/DMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/DMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1992

The origin and early evolution of nucleic acid polymerases

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

Lazcano, A.↗

NASA NTRS1995

The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could generate the RNA metabolic fossils we see today.

Dworkin, Jason P.↗

DOE OSTI2014

Structure and specificity of FEN-1 from Methanopyrus kandleri

DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Ultimately, we found both to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Base pairing, structural and functional insights into N 4 -methylcytidine (m 4 C) and N 4 ,N 4 -dimethylcytidine (m 4 2 C) modified RNA

The N 4 -methylation of cytidine (m 4 C and m 4 2 C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m 4 C and m 4 2 C 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 m 4 C 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 m 4 2 C 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 m 4 2 C 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 N 4 -position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

59 BASIC BIOLOGICAL SCIENCES↗