לוח שנה משולב

Department of Chemistry & Grossman Institute of Neuroscience and Quantitative Biology
The University of Chicago

DNA can be self-assembled into molecularly precise, well-defined, synthetic assemblies on the nanoscale, commonly referred to as designer DNA nanodevices. My lab creates synthetic, chemically responsive, DNA-based fluorescent probes. (1) In 2009 my lab discovered that these designer nanodevices could function as fluorescent reporters to quantitatively image ions in real time in living systems. (2,3) Until this innovation, it was not at all obvious whether such DNA nanodevices could function inside a living cell without being interfered with, or interfering with, the cells own networks of DNA control (4). In this talk I will discuss unpublished work on how we have expanded this technology from ion imaging (5,6) to now quantitatively imaging reactive species as well as enzymatic cleavage with sub-cellular spatial resolution in vivo.

References:

1. Chakraborty, K., et al., Nucleic acid based nanodevices in biological imaging. Ann. Rev. Biochem., 2016 85, 349-373.
2. Modi, S., et al. A DNA nanomachine that maps spatial and temporal pH changes in living cells. Nature Nanotechnology, 2009, 4, 325-330.
3. Modi, S., et al. Two DNA nanomachines map pH of intersecting endocytic pathways. Nature Nanotechnology, 2013, 8, 459-467.
4. Surana, S., et al. Designing DNA nanodevices for compatibility with the immune system of higher organisms. Nature Nanotechnology, 2015, 10, 741-747.
5. Saha, S., et al. A pH-insensitive DNA nanodevice quantifies chloride in organelles of living cells. Nature Nanotechnology, 2015, 10, 645-651.
6. Chakraborty, K., et al., High lumenal chloride in the lysosome is critical for lysosome function. eLife, 2017, 6, e28862.
7. Dan, K. et al., DNA nanodevices map enzymatic activity in vivo. 2018 (in revision).
8. Thekkan, S. et al A DNA-based fluorescent reporter maps HOCl dynamics in the maturing phagosome. 2018 (submitted)

Translesion DNA synthesis (TLS) overcomes arrest of replication forks at DNA lesions, by allowing synthesis across the damaged sites by specialized low-fidelity TLS DNA polymerases. This prevents double-strand breaks and genomic instability at the cost of increased point mutations. An siRNA screen performed in our lab in search for novel regulatory mammalian TLS genes, identified 17 novel TLS genes, one of which was PAPD7 (Poly (A) polymerase D7), a putative non-canonical poly(A) RNA polymerase. The biological role of PAPD7 is unknown yet.
We over-expressed and partially purified recombinant human PAPD7 and showed that it is indeed an adenylyltransferase. Measuring TLS across site-specific benzo[a]pyrene–G (BP-G), a major cigarette smoke DNA-adduct, we show that the down-regulation of PAPD7 decreased TLS across BP-G, and also decreased its mutagenicity. Further analysis showed that at least part of PAPD7 regulation of TLS is via its effect on monoubiquitination of PCNA (the DNA sliding clamp), a key step in TLS. RNA-seq analysis followed enrichment analysis showed that PAPD7 is involved in several biological functions including RNA metabolism, development, inflammation, signalling, cell cycle and DNA replication. Current studies are aimed at better understanding the molecular mechanism of TLS regulation by PAPD7.

Light-absorbing organic aerosol commonly termed as Brown carbon (BrC) is a significant contributor to radiative forcing of the Earth’s climate and also is of toxicological concern. Understanding the environmental effects of BrC, its sources, formation, and aging processes requires fundamental knowledge of its chromophores and characterization of their light-absorption properties. This seminar will highlight our recent analytical chemistry developments and applications in the area of molecular-level characterization of BrC that provided first insights into diverse composition and properties of its common chromophores. We present the chemical analysis of chromophores reported in a number of case studies of BrC materials associated with emissions from biomass burning and anthropogenic secondary organic aerosols. The results show that BrC chromophores include organic molecules with various structures, polarities, and volatilities. Understanding their chemical identity requires multi-modal analysis employing complementary separation and ionization approaches in combination with high resolution mass spectrometry. These studies allow assessment of BrC optical properties and relating them to fractional contributions from different classes of chromophores such as aromatic carboxylic acids, nitro-phenols; substituted, heterocyclic and pure polycyclic aromatic hydrocarbons.