Posted On Mar 31 2021

Question 1: The glucocorticoid receptor recognizes cortisol and other steroid hormones, which triggers its association with DNA and affecting the transcription of several genes. The glucocorticoid receptor (GRE) is upregulated in several types of cancer, notably various lymphomas. Accordingly, the GRE is an attractive target for treating cancer, as disruption of GRE binding to DNA would result in downregulation of the associated genes. (A) The consensus sequence for GRE recognition of DNA is provided below. Such DNA sequences may be selectively recognized by distamycin-inspired polyamides (see Lecture 3-1). Design a polyamide sequence using the three monomers (Py, Im, Hyp) below that could target the E-box consensus sequence (8 points). Hint: It may be useful to consult Dervan, Bioorg. Med. Chem., 2001, 9, 2215. GRE Recognition Sequence: 5′-GGTACA-3′ 3′-CCATGT-5′ (B) In order to assess whether or not the polyamide is successfully preventing GRE-regulated genes, a worthwhile experiment would be administration of the polyamide to lymphoma cells in culture and detection of transcribed RNA. In this case, reduction of transcripts of GRE-regulated genes may indicate successful disruption of its recognition of DNA. Briefly describe the steps necessary to determine whether GRE regulated RNA transcripts are affected by the designed polyamide, including any appropriate control(s). (7 points) Question 2: Staudinger ligations (the reaction between an azide and triphenylphosphine, see below) are a useful biorthogonal reaction. However, the reaction suffers from a slow second-order rate constant, often leading to incomplete reactions over timescales relevant (minutes to hours) for the covalent modification of biological molecules. One possibility to improve the reaction kinetic behavior is the development of catalysts capable of increasing the reaction rate. (A) Suppose you were tasked with generating an RNA catalyst (ribozyme) capable of catalyzing the Staudinger ligation reaction. Briefly describe the steps necessary to generate a ribozyme capable of catalyzing this reaction. For full credit, include a means of isolating catalytically active RNAs during your selection process. Hint: Recall the selection of a Diels-Alder ribozyme. (8 points) (B) Unfortunately, early attempts by your lab have failed to successfully crystallize or NMR characterize the threedimensional structure of the generated ribozyme catalyst. As an enterprising researcher, you are aware that at least a rough “solvent exposure,” map can be generated by reaction of the ribozyme with a SHAPE reagent like NMIA. Briefly describe how this process works. (7 points)

Question 3: Non-natural nucleobases have been explored as a potential means to alter the heritable information of living organisms or to potentially engineer novel function(s) into living cells. Consider the use of non-natural nucleobases in addressing the questions below. (A) Early research on the incorporation of non-natural nucleobases into DNA duplexes observed that including nucleobases into a short DNA duplex as a 5’-overhang led to an increase in melting temperature based on the base structure. Explain the trend in observed melting temperatures below, keeping in mind the physical interactions that govern biomolecule stability and that in regular dsDNA the bases stack on top of one another within each strand. (8 points) (B) Non-natural nucleobases have been proposed as a means to expand the genetic code of living organisms. This genetic code expansion can then be exploited for the incorporation of non-natural amino acids into proteins. Compare the utility of this method to (1) suppression of endogenous nonsense or “stop,” codons, such as the rarest nonsense codon UAG (Amber); (2) genetic code expansion through the translation of four-base codons (Chin, et al. Nat. Chem., 2014, 6, 393). Hint: Consider that the natural genetic code includes 4 nucleobases, with 3 in sequence per amino acid or 43 = 64 possible codons. Additionally, consider the potential permanency of “encoding,” non-natural functions and refer to the work of Romesberg, Nature, 2014, 509, 385. (7 points)

Question 4: The RNA interference (RNAi) pathway was discovered in C. elegans in 1993 and has since rapidly developed towards therapeutic applications, including the first approved RNAi therapeutic in August of 2018. Use your understanding of RNAi and its therapeutic applications to answer the questions below. (A) Compare and contrast the short interfering RNA (siRNA) and micro-RNA (miRNA) processes. In what ways are these two RNAi pathways similar? In what ways do they differ? (8 points) (B) Suppose you work for a pharmaceutical company developing an RNAi therapeutic. What considerations would you need to make to ensure proper selection of the desired single-stranded RNA (from a double-stranded RNA duplex) as the “guide,” RNA? What modifications would be made to the passenger strand in order to ensure that it is not used as the guide strand by RISC? How could you confirm the activity of your RNAi therapeutic in a cell culture model? (7 points)

Question 5: Gene editing technologies have significant implications for human health, particularly with respect to the potential elimination of heritable genetic disease. Use your knowledge of gene editing methods and DNA targeting to answer the questions below. (A) The use of CRISPR/Cas systems has greatly impacted genetic manipulation relative to previous gene-editing methods such as zinc fingers (ZFs) and transcription activator like effectors (TALEs). Compare/contrast CRISPR/Cas9 with ZFs and TALEs. Relative to these earlier systems, what is different about CRISPR/Cas9? What are the primary advantages of CRISPR/Cas9 for gene editing? (8 points) (B) Cas9 utilizes two domains to cleave target DNA. The RuvC domain (light blue, below) cleaves the unhybridized ssDNA, while the HNH domain (pink, below) is responsible for cleavage of the second DNA strand as its part of a RNA-DNA hybrid duplex (where RNA is responsible for recognizing the target DNA). (1) Based on the model below, propose two mutations that would inactivate Cas9 nuclease activity. (2) Propose a use for this nucleaseinactive “dead,” Cas9; you may consider fusion to an effector domain, but that may not be required for a practical use. (7 points)

Question 6: Below is information extracted from the crystal structure of E. coli tyrosyl-tRNA synthetase with tyrosine bound in the active site (PDB: 1X8X). Note: Active-site amino acids interacting with the bound amino acid are given by single-letter amino acid code and primary sequence position (e.g. D182 indicates aspartate at the 182nd position in the primary sequence). Using this structural information and your knowledge of protein translation, answer the questions below. Figure 1. Representations of tyrosine bound in the active site of E. coli tyrosyl-tRNA synthetase.

Left: Polar interaction map from PDB 1X8X from the RCSB protein databank. Blue dashed lines = hydrogen bonds; grey dashed lines = hydrophobic interactions. Middle: Space filling model of bound tyrosine (blue) from PDB 1X8X with nearby residues labeled. Right: Rotation of the space filling model of bound tyrosine (blue) at middle with nearby residues labeled. A. Suppose you wanted to engineer this synthetase to recognize propargyltyrosine (PpY, below) instead of tyrosine. What active site amino acid(s) would you mutate, and to which other amino acid? Briefly explain your choice(s). (7 points) B. Suppose you wanted to utilize your engineered synthetase to incorporate propargyltyrosine into a protein in response to a stop (TAG) codon during protein expression in mammalian cells in culture. Would this synthetase be suitable for use in mammalian cells? Why or why not? What other component(s) of the protein translational machinery would need to be engineered to accomplish your goal? (8 points)

Bonus Question (5 points): Peptide nucleic acids (PNAs) exhibit greater stability as heteroduplexes with DNA (i.e. PNADNA duplex) than does double-stranded DNA (i.e. DNA-DNA duplex). However, peptide nucleic acids lack charged groups, making them largely insoluble under near-physiological conditions in aqueous buffer. Provide (1) an explanation for the increased stability of PNA-DNA duplexes (hint: consider intermolecular forces). (2) Additionally, propose modification(s) of the PNA scaffold that could increase solubility without drastically reducing duplex stability.

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