Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and non-cancerous cells.

Promoters often contain asymmetric G- and C-rich strands, in which the cytosines are prone to epigenetic modification via methylation (5-mC) and 5-hydroxymethylation (5-hmC). These sequences can also form four-stranded G-quadruplex (G4) or i-motif (iM) secondary structures. Although the requisite sequences for epigenetic modulation and iM/G4 formation are similar and can overlap, they are unlikely to coexist. Despite 5-hmC being an oxidization product of 5-mC, the two modified bases cluster at distinct loci. This study focuses on the intersection of G4/iM formation and 5-hmC modification using the vascular endothelial growth factor (VEGF) gene promoter's CpG sites and examines whether incorporation of 5-hmC into iM/G4 structures had any physicochemical effect on formation, stability, or recognition by nucleolin or the cationic porphyrin, TMPyP4. No marked changes were found in the formation or stability of iM and G4 structures; however, changes in recognition by nucleolin or TMPyP4 occurred with 5-hmC modification wherein protein and compound binding to 5-hmC modified G4s was notably reduced. G4/iM structures in the VEGF promoter are promising therapeutic targets for antiangiogenic therapy, and this work contributes to a comprehensive understanding of their governing principles related to potential transcriptional control and targeting.

B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine–cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the p K a of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1–30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine–iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo.

molecular determinants of translation speed are distributed randomly across the mRNA transcripts and that this distribution is generated from a Poisson point process. As a consequence, the Law of Large Numbers is followed and for any given transcript fast-translating segments are canceled out by a similar number of slow-translating segments resulting a constant average codon translation rate between different transcripts. This means that a protein's average synthesis time can be accurately predicted based solely on the corresponding gene length, provided the average codon translation speed is known. Thus, although evolution has biased codon usage between different genes in an organism, there is still a large degree of randomness associated with how individual codon translation speeds have been distributed. These results also provide an explanation for the observation from ribosome profiling that different transcripts have the same average codon translation speed in mouse-stem cells. 344-Pos Board B109The ribosome is a large ribonucleoprotein complex that is responsible for translation of proteins in all living organisms. Accommodation is a key conformational change during transfer RNA (tRNA) selection that allows for each molecule to fully associate with the ribosome. During tRNA accommodation, there are large-scale fluctuations in the L11 stalk and the associated protein. Experimental and theoretical work has shown that the L11 protein and RNA exhibit dynamic behavior, where their precise position can significantly affect the dynamics of elongation. To explore the impact of L11 stalk mobility on the kinetics of aa-tRNA accommodation, we used molecular dynamics simulations with a simplified model to evaluate the free-energy as a function of aa-tRNA position. We find that as the flexibility of L11 is decreased, the position of the free-energy minima is robust. In contrast, the magnitude of the free-energy barrier for accommodation depends on the precise scale of L11 fluctuations. We find that increasing the rigidity of the L11 stalk can destabilize the A/T ensemble. In addition to the close relationship between L11 mobility and the free-energy landscape of accommodation, the direction of L11 stalk movement is also correlated with aa-tRNA displacements. To probe the collective motion of the L11 stalk, we performed Principal Component Analysis on the simulated trajectories. When aa-tRNA is first delivered to the ribosome, the stalk moves away from the tRNA and adopts extended configurations. It then relaxes during the accommodation process. Together, these calculations reveal a correlation between fluctuations in the L11 stalk and the dynamics of aa-tRNA during accommodation. This provides a quantitative foundation for interpreting experimental measures of accommodation and suggests how the dynamics of the L11 stalk may contribute to aa-tRNA proof-reading and accommodation. 345-Pos Board B110Simulating Movement of the Ribosome Head during Translocation Intrasubunit head movement has been identified as an essential m...

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