POLD1 DEDD Motif Mutation Confers Hypermutation in Endometrial Cancer and Durable Response to Pembrolizumab.

BACKGROUND: Mutations in the DNA polymerase delta 1 (POLD1) exonuclease domain cause DNA proofreading defects, hypermutation, hereditary colorectal and endometrial cancer, and are predictive of immunotherapy response. Exonuclease activity is carried out by two magnesium cations, bound to four highly conserved, negatively charged amino acids (AA) consisting of aspartic acid at amino acid position 316 (p.D316), glutamic acid at position 318 (p.E318), p.D402, and p.D515 (termed DEDD motif). Germline polymorphisms resulting in charge-discordant AA substitutions in the DEDD motif are classified as variants of uncertain significance (VUSs) by laboratories and thus would be considered clinically inactionable. We hypothesize this mutation class is clinically pathogenic. METHODS: A review of clinical presentation was performed in our index patient with a POLD1(p.D402N) heterozygous proband with endometrial cancer. Implications of this mutation class were evaluated by a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)-guided systematic review, in silico analysis with orthogonal biochemical confirmation, and whole-exome and RNA sequencing analysis of the patient's tumor and engineered cell lines. RESULTS: Our systematic review favored a Mendelian disease mutation class associated with endometrial and colorectal cancers. In silico analysis predicted defective protein function, confirmed by biochemical assay demonstrating loss of nuclease activity. A POLD1-specific mutational signature was found in both the patient's tumor and POLD1(p.D402N) overexpressing cell. Furthermore, paired whole-exome/transcriptome analysis of endometrial tumor demonstrated hypermutation and T cell-inflamed gene expression profile (GEP), which are joint predictive biomarkers for pembrolizumab. Our patient showed a deep, durable response to immune checkpoint inhibitor (ICI). CONCLUSION: Charge-discordant AA substitution in the DEDD motif of POLD1 is detrimental to DNA proofreading and should be reclassified as likely pathogenic and possibly predictive of ICI sensitivity.

Okazaki fragment maturation: DNA flap dynamics for cell proliferation and survival.

Unsuccessful processing of Okazaki fragments leads to the accumulation of DNA breaks which are associated with many human diseases including cancer and neurodegenerative disorders. Recently, Okazaki fragment maturation (OFM) has received renewed attention regarding how unprocessed Okazaki fragments are sensed and repaired, and how inappropriate OFM impacts on genome stability and cell viability, especially in cancer cells. We provide an overview of the highly efficient and faithful canonical OFM pathways and their regulation of genomic integrity and cell survival. We also discuss how cells induce alternative error-prone OFM processes to promote cell survival in response to environmental stresses. Such stress-induced OFM processes may be important mechanisms driving mutagenesis, cellular evolution, and resistance to radio/chemotherapy and targeted therapeutics in human cancers.

Structure-specific nucleases: role in Okazaki fragment maturation.

Proper function of structure-specific nucleases is key for faithful Okazaki fragment maturation (OFM) process completion. Deregulation of such nucleases leads to aberrant OFM and causes a spectrum of mutations, some of which may confer survival outcomes under specific stresses and serve as attractive targets for therapeutic intervention in human cancers.

Selection of a self-cleaving ribozyme activated in a chemically and thermally denaturing environment.

A new self-cleaving ribozyme was obtained from in vitro selection, displaying site-specific cleavage activity under denaturing conditions, such as high temperatures up to 95 °C, and denaturing solvents (20 M formamide). Adding salt such as Mg2+ also inhibited its activity. The conserved ribozyme sequence was found in the genome of several extremophiles, suggesting its potential biological relevance. This study provides an example of a ribozyme working under exotic conditions, which may expand the application of ribozymes in non-biological environments.

Freezing-Assisted Conjugation of Unmodified Diblock DNA to Hydrogel Nanoparticles and Monoliths for DNA and Hg2+ Sensing.

Acrydite-modified DNA is the most frequently used reagent to prepare DNA-functionalized hydrogels. Herein, we show that unmodified penta-adenine (A5 ) can reach up to 75 % conjugation efficiency in 8 h under a freezing polymerization condition in polyacrylamide hydrogels. DNA incorporation efficiency was reduced by forming duplex or other folded structures and by removing the freezing condition. By designing diblock DNA containing an A5 block, various functional DNA sequences were attached. Such hydrogels were designed for ultrasensitive DNA hybridization and Hg2+ detection, with detection limits of 50 pM and 10 nM, respectively, demonstrating the feasibility of using unmodified DNA to replace acrydite-DNA. The same method worked for both gel nanoparticles and monoliths. This work revealed interesting reaction products by exploiting freezing and has provided a cost-effective way to attach DNA to hydrogels.

Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology.

Since the initial discovery of ribozymes in the early 1980s, catalytic nucleic acids have been used in different areas. Compared with protein enzymes, catalytic nucleic acids are programmable in structure, easy to modify, and more stable especially for DNA. We take a historic view to summarize a few main interdisciplinary areas of research on nucleic acid enzymes that may have broader impacts. Early efforts on ribozymes in the 1980s have broken the notion that all enzymes are proteins, supplying new evidence for the RNA world hypothesis. In 1994, the first catalytic DNA (DNAzyme) was reported. Since 2000, the biosensor applications of DNAzymes have emerged and DNAzymes are particularly useful for detecting metal ions, a challenging task for enzymes and antibodies. Combined with nanotechnology, DNAzymes are key building elements for switches allowing dynamic control of materials assembly. The search for new DNAzymes and ribozymes is facilitated by developments in DNA sequencing and computational algorithms, further broadening our fundamental understanding of their biochemistry.

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation.

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.

An in Vitro-Selected DNAzyme Mutant Highly Specific for Na+ under Slightly Acidic Conditions.

Sodium is one of the most common metal ions in biology; however, DNA-based sodium probes have only been reported recently. A Na+ -specific RNA-cleaving DNAzyme named NaA43 is active with Na+ alone. In this work, we were using Co(NH3 )63+ as the intended metal cofactor for in vitro selection, but obtained a mutant of the NaA43 DNAzyme. The mutant was named NaH1, and differs from NaA43 by only two nucleotides. NaA43 has an optimal pH of 7.0, whereas the optimal pH for NaH1 is 6.0. This difference might be due to our selection having been performed at pH 6.0. NaH1 also displays an excellent selectivity for sodium relative to other competing monovalent ions, as well as a fast catalytic rate of (0.11±0.01) min-1 with 50 mm Na+ . At low Na+ concentrations, the selected DNAzyme exhibited a higher cleavage rate than NaA43 and thus a tighter apparent Kd of (12.0±1.6) mm Na+ . Furthermore, the NaH1 DNAzyme was engineered into a fluorescent Na+ biosensor by attaching a fluorophore/quencher pair to the DNAzyme with a detection limit of 223 µm Na+ . Preliminary work on detection of Na+ in serum was demonstrated as well. This study provides a useful mutant that works in a slightly acidic environment, which might be useful for sensing Na+ in acidic in vivo environments.

Misfolding of a DNAzyme for ultrahigh sodium selectivity over potassium.

Herein, the excellent Na+ selectivity of a few RNA-cleaving DNAzymes was exploited, where Na+ can be around 3000-fold more effective than K+ for promoting catalysis. By using a double mutant based on the Ce13d DNAzyme, and by lowering the temperature, increased 2-aminopurine (2AP) fluorescence was observed with addition of both Na+ and K+. The fluorescence increase was similar for these two metals at below 10 mM, after which K+ took a different pathway. Since 2AP probes its local base stacking environment, K+ can be considered to induce misfolding. Binding of both Na+ and K+ was specific, since single base mutations could fully inhibit 2AP fluorescence for both metals. The binding thermodynamics was measured by temperature-dependent experiments revealing enthalpy-driven binding for both metals and less coordination sites compared to G-quadruplex DNA. Cleavage activity assays indicated a moderate cleavage activity with 10 mM K+, while further increase of K+ inhibited the activity, also supporting its misfolding of the DNAzyme. For comparison, a G-quadruplex DNA was also studied using the same system, where Na+ and K+ led to the same final state with only around 8-fold difference in Kd. This study provides interesting insights into strategies for discriminating Na+ and K+.

Bromide as a Robust Backfiller on Gold for Precise Control of DNA Conformation and High Stability of Spherical Nucleic Acids.

Functionalization of a gold surface with DNA is often complicated by kinetic traps from unintended DNA base adsorption. Herein, we communicate that Br- serves as a robust backfilling agent displacing selected DNA bases on gold. Traditional thiol backfillers are too strong, while even 300 mM Br- is well tolerated. Conjugates prepared with Br- hybridize 10-fold faster and resist DNA release with better colloidal stability yielding highly sensitive probes. From colorimetric and Raman assays, adsorption affinity ranks as F- < T ≈ Cl- < C < G ≈ Br- < A < I-, allowing Br- to displace nonpoly-A sequences from gold. This well-controlled biointerface will impact biosensing, drug delivery, and directed assembly of nanomaterials.

DNA Adsorption by ZnO Nanoparticles near Its Solubility Limit: Implications for DNA Fluorescence Quenching and DNAzyme Activity Assays.

Zinc oxide (ZnO) is a highly important material, and Zn(2+) is a key metal ion in biology. ZnO and Zn(2+) interconvert via dissolution and hydrolysis/condensation. In this work, we explore their interactions with DNA, which is important for biointerface, analytical, and bioinorganic chemistry. Fluorescently labeled DNA oligonucleotides were adsorbed by a low concentration (around 5 µg/mL) of ZnO nanoparticles, near the solubility limit. Right after mixing, fluorescence quenching occurred, indicating DNA adsorption. Then, fluorescence recovered, attributable to ZnO dissolution. The dissolution rate followed A5 > T5 > C5. Dissolution was slower with longer DNA. The adsorption affinity was also measured by a displacement assay to be G5 > C5 > T5 > A5, suggesting that tightly adsorbed DNA can retard ZnO dissolution. Electrostatic interactions are important for DNA adsorption because ZnO is positively charged at neutral pH, and a high salt concentration inhibits DNA adsorption. Next, in situ formation of ZnO from Zn(2+) was studied. First, titrating Zn(2+) into a fluorescently labeled oligonucleotide at pH 7.5 resulted in an abrupt fluorescence quenching beyond 0.2 mM Zn(2+). At pH 6, quenching occurred linearly with the Zn(2+) concentration, suggesting the effect of Zn(2+) precipitation at pH 7.5. Second, a Zn(2+)-dependent DNA-cleaving DNAzyme was studied. This DNAzyme was inhibited at higher than 2 mM Zn(2+), attributable to Zn(2+) precipitation and adsorption of the DNAzyme. This paper has established the interplay between DNA, Zn(2+), and ZnO. This understanding can avoid misinterpretation of DNA assay results and adds knowledge to DNA immobilization.