Biophysical and biochemical characteristics of G4C2 DNA repeats

Amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD) sta dve nevrodegenerativni bolezni, ki delita nevropatološke, klinične in genetske značilnosti. Pokazalo se je, da je razširjena heksanukleotidna ponovitev G4C2 znotraj gena C9orf72, najpogostejši genetski vzrok ALS in pomembno prisotna pri FTD. Zdravi posamezniki imajo do 23 ponovitev G4C2. Število pri bolnikih z ALS ali FTD znaša tudi do več 1000. Mehanizem z mutacijo v genu C9orf72 posredovane nevrodegeneracije ni popolnoma znan. V magistrski nalogi smo sprva z opazovanjem spremembe mobilnosti DNA na agarozni gelski elektroforezi (AGE) preučevali vplive temperature, pH in ionskih zvrsti na različno dolga zaporedja s ponovitvami G4C2 ter opazili očitne razlike v pogojih denaturacije med konstrukti, ki vsebujejo ponovitve G4C2 ter kontrolnimi konstrukti, ki so naključna zaporedja DNA, podobne dolžine kot preiskovani konstrukti. Razlike med kontrolnim zaporedjem in zaporedjem G4C2 smo pripisali GC bogatemu zaporedju. Pogoje denaturacije verig pridobljene na AGE smo potrdili z mikroskopijo na atomsko silo (AFM). Z gvanini bogata zaporedja tvorijo G-kvadruplekse, nekanonične sekundarne strukture sestavljene iz planarnih tetramernih kvartetov iz štirih zaporednih gvaninov. Doslej so pokazali tvorbo G-kvadrupleksov znotraj zaporedij, ki vsebujejo do 5 ponovitev G4C2. Vendar pa ostaja izziv potrditi tvorbo le-teh znotraj molekul DNA, ki vsebujejo večje število ponovitev. Zaporedja, ki vsebujejo 48 ponovitev G4C2, imajo večji biološki pomen, kot zaporedja z manjšim številom (< 24). Zato smo se v magistrski nalogi lotili dokazovanja tvorbe G-kvadrupleksov v zaporedju DNA z 48 ponovitvami G4C2 s CD-spektroskopijo ter detekcijo intenzitete fluorescence fluorescenčnega barvila, saj se mu ob vezavi na G-kvadrupleks le-ta poveča. Detekcija DNA G-kvadrupleksov s fluorescenčnim barvilom je dala obetavne rezultate, ki nakazujejo na prisotnost teh struktur v zaporedju s ponovitvami G4C2. Posneti CD-spektri so pokazali spremembo strukture DNA po denaturaciji tako pri kontrolnem zaporedju DNA kot pri zaporedju DNA s ponovitvami G4C2. Dodatno so CD-spektri zaporedja G4C2 vsebovali vrhove, ki kažejo na prisotnost G-kvadrupleksov znotraj ponovitev G4C2, medtem ko pri kontrolnem zaporedju teh vrhov nismo zaznali. Ker je bilo do danes narejenih veliko študij vpliva RNA G-kvadrupleksov na razvoj ALS ter FTD, smo detekcijo s fluorescenčnim barvilom izvedli tudi na RNA in zaznali prisotnost G-kvadrupleksov v G4C2 zaporedju RNA. Nazadnje smo pripravili z biotinom označene enojne verige DNA s ponovitvami G4C2, ki jih bomo v nadaljnjem delu uporabili za DNA test ˝pull down˝ z namenom identifikacije proteinov, ki se vežejo na omenjene konstrukte. Z namenom označevanja konstruktov DNA z biotinom smo optimizirali reakcijo PCR in uspešno pomnožili zaporedje s 100 % deležem GC.Amyotrophic lateral sclerosis (ALS) in frontotemporal dementia (FTD) are two types of neurodegenerative diseases, that share neuropathological, clinical and genetic properties. It was shown that hexanucleotide repeat expansion in gene C9orf72, is the most common genetic cause of ALS and significantly present also in FTD. Healthy individuals normally carry not more than 23 G4C2 repeats. However, the number of repeats in ALS or FTD patients can be more than 1000. The neurodegeneration mechanism mediated by a mutation in the C9orf72 gene is not fully understood. In master thesis, we started with observations of change in mobility of DNA with agarose gel electrophoresis (AGE) and through this we studied the effects of temperature, pH and different ions on sequences with G4C2 repeats of different length and observed noticeable differences in conditions of denaturation between constructs containing G4C2 repeats and control constructs, which are random DNA sequences of similar length to studied constructs. Differences between the control and G4C2 sequences were attributed to the GC-rich sequence. We confirmed denaturation conditions obtained on AGE with atomic force microscopy (AFM). Sequences that are rich in guanine form G-quadruplexes, non-canonical secondary structures made of planar tetramer quartet units from four consecutive guanines. So far, the formation of G-quadruplexes has been shown inside sequences containing up to 5 G4C2 repeats. However, it remains a challenge to confirm the presence of G-quadruplexes within DNA molecules containing a higher number of repeats. Sequences containing 48 G4C2 repeats are of greater biological relevance than sequences with a lower number of repeats (< 24). Hence, in master thesis we aimed to prove a formation of G-quadruplexes inside the DNA sequence containing 48 G4C2 repeats with CD spectroscopy and detection of fluorescence intensity of fluorescent dye, since after its binding to G-quadruplex the fluorescence intensity increases. The fluorescent dye gave promising results suggesting the presence of these structures in DNA sequence with G4C2 repeats. CD spectrums showed a shift in the DNA structure after denaturation of control sequence as well as within sequence with G4C2 repeats. Moreover, CD spectrums of G4C2 contained peaks, that point to the presence of G-quadruplexes inside the G4C2 repeats, while in spectrums of control sequence we were not able to locate them. Because of all the studies of a contribution of RNA G-quadruplexes to development of ALS and FTD that were made by now, we also performed a detection of these secondary structures with fluorescent dye using RNA molecules and were able to detect a presence of G-quadruplexes within G4C2 RNA sequence. Lastly, we prepared biotin-labeled single strands of DNA containing G4C2 repeats, that we will use in our further work for DNA ˝pull down˝ test in order to identify proteins, which bind to described constructs. With the purpose of labeling DNA constructs with biotin, we optimized PCR reaction and successfully amplified the sequence that is 100 % GC