KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

The oncogene MYC has key roles in transcription, proliferation, deregulating cellular energetics, and more. Modulating the expression or function of the MYC protein is a viable therapeutic goal in an array of cancer types, and potential inhibitors of MYC with high specificity and selectivity are of great interest. In cancer cells addicted to their aberrant MYC function, suppression can lead to apoptosis, with minimal effects on non-addicted, non-oncogenic cells, providing a wide therapeutic window for specific and efficacious anti-tumor treatment. Within the promoter of MYC lies a GC-rich, G-quadruplex (G4)-forming region, wherein G4 formation is capable of mediating transcriptional downregulation of MYC. Such GC-rich regions of DNA are prime targets for regulation with Polypurine Reverse Hoogsteen hairpins (PPRHs). The current study designed and examined PPRHs targeting the G4-forming and four other GC-rich regions of DNA within the promoter or intronic regions. Six total PPRHs were designed, examined in cell-free conditions for target engagement and in cells for transcriptional modulation, and correlating cytotoxic activity in pancreatic, prostate, neuroblastoma, colorectal, ovarian, and breast cancer cells. Two lead PPRHs, one targeting the promoter G4 and one targeting Intron 1, were identified with high potential for further development as an innovative approach to both G4 stabilization and MYC modulation.

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

KRAS is a GTPase involved in the proliferation signaling of a number of growth factors, such as epidermal growth factor. The protein is mutated or upregulated in an array of cancers, including pancreatic and ovarian cancers, where it correlates with more aggressive and chemoresistant disease. Within the promoter of KRAS lies a G-rich region capable of forming the higher order non-canonical DNA structure, the G-quadruplex (G4). Stabilization of G4 formation in the mid-region of the KRAS promoter downregulates transcription and facilitates decreased growth and chemo-sensitization of pancreatic and ovarian cancer cells with aberrant KRAS signaling. Our collaborative team designed Polypurine Reverse Hoogsteen (PPRH) hairpin oligonucleotides that establish Watson Crick bonds with the pyrimidine strand within the KRAS-mid-G4-forming region to enhance G4 formation, downregulate KRAS transcription and mediate an anti-proliferative effect. In particular, two PPRHs were designed to interact with the 3’ and 5’ ends of the G4-forming region, along with two correlating W:C, and one scramble, controls. The binding of these oligonucleotides to the G4-forming region was verified by gel electrophoresis, and the cell activity was examined in vitro both by luciferase and in pancreatic and ovarian cancer cells. Using a luciferase construct driven by the KRAS promoter, only the PPRHs, and none of the control oligonucleotides, significantly decreased activity. Cytotoxicity experiments in KRAS dependent and independent cell lines demonstrated that PPRHs, but not control oligonucleotides, selectively suppressed proliferation only in the dependent pancreatic and ovarian cancer cell lines. Correlation of the cytotoxic effects of these potential therapeutic PPRHs with transcriptional downregulation is under investigation. PPRH also demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound in KRAS-dependent pancreatic cells, and sensitization to standard chemotherapeutic regimens is also being studied. These designed PPRH oligonucleotides selectively stabilize G4 formation within the KRAS mid promoter region, and represent an innovative approach both for G4-stabilization and KRAS modulation with potential for development into novel therapeutics. Citation Format: Alexandra Maria Psaras, Simonas Valiuska, Veronique Noe, Carlos J. Ciudad, Tracy A. Brooks. Facilitating G-quadruplex formation in the KRAS promoter with polypurine reverse Hoogsteen oligonucleotides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 675.