ARRSProjekti/2018/Fazni prehodi v sistemih periodičnih nukleotidnih ekspanzij povezanih z nevrodegenerativnimi boleznimi

Fazni prehodi v sistemih periodičnih nukleotidnih ekspanzij povezanih z nevrodegenerativnimi boleznimi

Nazaj na seznam za leto 2018

Oznaka in naziv projekta

J7-9399 - Fazni prehodi v sistemih periodičnih nukleotidnih ekspanzij povezanih z nevrodegenerativnimi boleznimi J7-9399 Phase transitions in systems of nucleotide repeat expansions associated with neurodegenerative diseases

Bibliografija projekta

SICRIS

Logotipi ARRS in drugih sofinancerjev

PROJEKTNA SKUPINA

Vodja projekta: prof. dr. Irena DREVENŠEK OLENIK

Sodelujoče raziskovalne organizacije:

1. Institut »Jožef Stefan«;

2. Univerza v Ljubljani, Fakulteta za matematiko in fiziko;

3. Kemijski inštitut.

Raziskovalci:

- Nerea SEBASTIAN UGARTECHE

PREDSTAVITEV PROBLEMA

Več kot 30 neozdravljivih nevrodegenerativnih bolezni je povezanih z genetskimi mutacijami, za katere so značilne ekspanzije kratkih nukleotidnih ponovitev. Njihov enotni patološki razvoj poteka preko faznega prehoda primarnih RNK transkriptov iz običajnega tekočinskega stanja v stanje kondenziranih skupkov, znanih kot nenadzorovane jedrne kapljice (tudi jedrna žarišča, granule RNK, itd.), katerih nastanek ima po navadi negativen, izjemoma pa tudi zaščitni vpliv na celične funkcije. Kondenzirane kapljice interagirajo z RNK-vezavnimi proteini, kar ima za posledico zaviranje naravnih funkcij teh proteinov in posledično povzroči usodne napake pri obdelavi in transkripciji RNK. Nedavne študije so pokazale, da nastajanje kondenziranih kapljic poteka ne le znotraj bioloških celic, temveč tudi v in-vitro sistemih. S tem se odpira možnost raziskovanja z njimi povezanih faznih prehodov in faznih separacijskih procesov z novega vidika, tj. z metodami, ki se običajno uporabljajo za preučevanje tovrstnih pojavov na področju fizike.

Molekularni mehanizmi, ki uravnavajo oblikovanje jedrnih kapljic in njihovo občutljivost na dolžino zaporedja, še vedno niso poznani. Vendar pa je bilo nedavno predlagano, da imajo pri njihovi tvorbi promocijsko vlogo nekonvencionalni mehanizmi zvijanja RNK, zlasti G-kvadrupleksno zvitje. Zato je oblikovanje kvadrupleksa DNK/RNK morebiti splošno pomemben vmesni korak na poti k razvoju motenj povezanih z nukleotidnimi ekspanzijami. Ključna odprta vprašanja so:

· Kako dolžina ponavljajočih se nukleotidnih zaporedij vpliva na njihovo organizacijo v strukture G-kvadrupleksov in druge strukture višjega reda?

· Zakaj, kako in pri katerih pogojih zgoraj omenjene strukture spodbujajo fazne prehode in fazno separacijo v kondenzirane jedrne kapljice?

· Kakšne so fizikalna narava in fizikalne lastnosti termodinamske faze, ki tvori kapljice?

· Kakšne so možnosti za regulacijo nastajanja in razpada kapljic z zunanjimi dodatki ali stimuli?

CILJI PROJEKTA

Za različno dolge nukleotidne sekvence bomo določili fazni diagram njihovih termodinamskih faz in določili osnovne fizikalne lastnosti teh faz. Ker pričakujemo pojav kromoničnih tekoče-kristalnih faz, bodo primarne preiskovane lastnosti tekočekristalni ureditveni red, optična in magnetna anizotropija, vizko-elastične konstante, periodičnost kiralne modulacije, itd. Preučili bomo tudi učinke dodajanja različnih ionov (npr. Na⁺, K⁺, Mg²⁺), zgoščevalcev (npr. PEG), komplementarnih sekvenc, G-kvadrupleksnih ligandov (npr. porfirini) in nekaterih beljakovin z znano težnjo, da se vežejo na granule RNK (npr. SF2, hnRNP-H, HuR). Projekt bo osredotočen na sledeče glavne cilje:

· Strukturna karakterizacija spontano formiranih visoko-organiziranih struktur (G-kvadrupleksi, G-žice, itd.) na osnovi ponavljajočih se nukleotidnih zaporedij,

· Sistematična določitev različnih faz in faznih prehodov v raztopinah ponavljajočih se nukleotidnih zaporedij različnih dolžin – določitev faznega diagrama,

· Določitev učinka različnih dodatkov in zunanjih stimulov na process fazne separacije in tvorbo kondenziranih kapljic.

Osnovni podatki sofinanciranja so dostopni na spletni strani. Povezava na SICRIS.

Faze projekta v celotnem obdobju trajanja na mesečni (m) bazi za obdobje 0-36 m:

Faza 1: Formiranje struktur višjega reda
Cilj: Strukturna karakterizacija spontano formiranih visoko-organiziranih struktur (G-kvadrupleksi, G-žice, itd.) na osnovi ponavljajočih se nukleotidnih zaporedij različnih dolžin
Naloge:	N1.1. – Priprava materialov in vzorcev (m1-m18), N1.2. – Karakterizacija spontano-formiranih visoko-organiziranih struktur v raztopini (m6-m24), N1.3. - Karakterizacija spontano-formiranih visoko-organiziranih struktur v strjenih filmih (m6-m24).
Pričakovani rezultati:	R1.1. – Raztopine in tanki filmih različnih ponavljajočih se sekvenc DNK in RNK R1.2. – Karakterizacija G-quadrupleksnih in drugih više organiziranih struktur v raztopinah z različnimi eksperimentalnimi tehnikami. R1.3. - Karakterizacija G-quadrupleksnih in drugih više organiziranih struktur v tankoplastnih nanosih s pomočjo AFM tehnike.
Diseminacija:	Znanstveni članki, konferenčni prispevki, študentski seminarji in diplomske ter magi-strske naloge, objave in novice v lokalnih časopisih, na spletnih portalih, radiu in TV.

Faza 2: Faze in fazni prehodi
Cilj: Sistematična določitev različnih faz in faznih prehodov v raztopinah ponavljajočih se nukleotidnih zaporedij različnih dolžin – določitev faznega diagrama
Naloge:	N2.1. – Klasifikacija različnih faz in faznih prehodov (m12-m30), N2.2. – Določitev fizikalnih lastnosti različnih faz (m12-m30), N2.3. – Teoretični model faznih prehodov (m1-m18).
Pričakovani rezultati:	R2.1. – Fazni diagram različnih faz za izbrane ponavljajoče se sekvence. R2.2. – Poznavanje osnovnih fizikalnih lastnosti N* kromatične TK faze za izbrane ponavljajoče se sekvence. R2.3. – Konstrukcija modela za I-N* fazni prehod v odvisnosti od dolžine sekvence.
Diseminacija:	Znanstveni članki, konferenčni prispevki, študentski seminarji in diplomske ter magi-strske naloge, objave in novice v lokalnih časopisih, na spletnih portalih, radiu in TV.

Faza 3: Učinek aditivov in stimulusov
Cilj: Določitev učinka različnih aditivov in zunanjih stimulusov na proces fazne separacije in tvorbo kondenziranih kapljic
Naloge:	Naloga 3.1. - Raziskave vpliva dodatka (bio)kemičnih aditivov na zgoščene faze (m18-m36), Naloga 3.2. – Raziskave učinka zunanjih stimulosov na zgoščene faze (m18-m36), Naloga 3.3. – Teoretični modeli interakcije različnih faz z aditivi in stimulusi (m18-m36).
Pričakovani rezultati:	R3.1. – Karakterizacija vpliva različnih dodatkov na fazni prehod I-N. R3.2. – Karakterizacija vpliva magnetnega polja in optičnega obsevanja na fazni prehod I-N. R3.3. – Konstrukcija teoretičnega modela za opis vpliva različnih dodatkov oz. stimulusov na fazni prehod I-N*.
Diseminacija:	Znanstveni članki, konferenčni prispevki, študentski seminarji in diplomske ter magi-strske naloge, objave in novice v lokalnih časopisih, na spletnih portalih, radiu in TV.

VMESNO POROČILO (maj 2020)

Večina raziskav v začetnem obdobju izvajanja projekta je bila osredotočena na raziskave mehanizmov zvijanja in agregacijskih procesov v relativno kratkih z gvanozinom bogatih oligonukleotidih DNK. To so raziskave, ki smo jih načrtovali predvsem v prvem delovnem sklopu projekta (DS1 – Formiranje struktur višjega reda). Pri delu smo sodelovali zlasti z raziskovalno skupino prof. S. Masiera iz Univerze v Bolonji v Italiji. Del aktivnosti je potekal tudi v sodelovanju s Fakulteto za naravoslovje in matematiko Univerze v Mariboru.

CILJ 1: Strukturna karakterizacija spontano formiranih visoko-organiziranih struktur (Gkvadrupleksi, G-žice, itd.) na osnovi ponavljajočih se nukleotidnih zaporedij različnih dolžin.

Za doseganje navedenega cilja smo izvedli obširno analizo procesov zvijanja z gvanozinom bogatih sekvenc DNK v G-kvadrupleksne strukture v raztopinah in v strjenih filmih. Poskuse v raztopinah izvajamo pod pogoji, ki so podobni (ali ustrezajo) fiziološkim pogojem. S tehniko dinamičnega sipanja svetlobe (DLS) v kombinaciji z mikroskopijo na atomsko silo (AFM) smo raziskovali mehanizme zvijanja v G-kvadruplekse in njihovo agregacijo v G-žice pri sekvencah (G4C2)xn za n=1, 2 in 4 [1,2,3]. Analizirali smo tudi nekatere druge sorodne z gvanozinom-bogate sekvence DNK s podobno dolžino [4-6], in sekvence humanega papiloma virusa [7], pri čemer je glavnino raziskav predstavljala NMR spektroskopija. Poleg tega raziskujemo tudi interakcijske lastnosti med različnimi derivati gvanozina in derivati drugih nukleobaz. Tovrstne raziskave so fokusirane na problematiko tvorbe in zaznavanja baznega parjenja v dvodimenzionalnih tankih plasteh na vodni površini [8]. Proučujemo pa tudi spontano-formirane visoko-organizirane strukture v strjenih filmih prenešenih iz vodne površine na trdne substrate [9,10].

CILJ 2: Sistematična določitev različnih faz in faznih prehodov v raztopinah ponavljajočih se nukleotidnih zaporedij različnih dolžin – določitev faznega diagrama.

Začeli smo tudi z raziskavami specifičnih mehanizmov zvijanja dolgih verig DNK z vgrajenimi sekvencami (G4C2)x48. Z mikroskopijo na atomsko silo (AFM) smo izvedli analizo agregacijskih lastnosti tovrstnih DNK plazmidov v primerjavi z analognimi plazmidi z vgrajeno enako dolgo naključno sekvenco. Proučevali smo tudi denaturacijske in renaturacijske mehanizme v navedenih sistemih [11, 12]. Nadaljnje raziskave na tem področju so v teku. Pripravljamo tudi prvi znanstveni članek iz navedene tematike.

CILJ 3: Določitev učinka različnih aditivov in zunanjih stimulusov na proces fazne separacije in tvorbo kondenziranih kapljic.

Za realizacijo navedenega cilja je potrebno najprej zaključiti prvi dva delovna sklopa projekta, zato izvedbo aktivnosti povezanih z njim načrtujemo v zadnjem letu izvajanja projekta. Med raznovrstnimi zunanjimi stimuli nas bo zanimal tudi vpliv UV svetlobe na procese fazne separacije. Za ta namen smo nedavno izdelali osvetljevalni sistem z dvema vrstama LED izvorov

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15. RUPP, Romano Anton, SEBASTIÁN UGARTECHE, Nerea, ŠTURM, Nika, DREVENŠEK OLENIK, Irena, MALNAR, Mirjana, ŽBOGAR, Karmen, ROGELJ, Boris, SPINDLER, Lea. Selfassembly of G4C2G4C2 repeats into G-quadruplexes: from short oligonucleotides to long DNA molecules. V: The 7th International Meeting on Quadruplex Nucleic Acids, (2019G4), September 6-9, 2019, Changchun, China. Changchun: [s. n.]. 2019, str. [197-198], ilustr. [COBISS.SI-ID 3355492].

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Reference - SICRIS

PRELIMINARNI REZULTATI

Slika 1

PROJECT TEAM

Project leader: prof. dr. Irena DREVENŠEK OLENIK

Participating research institutions:

1. J. Stefan Institute;

2. University of Ljubljana, Faculty of Mathematics and Physics;

3. National Institute of Chemistry.

RESEARCHERS:

- Nerea SEBASTIAN UGARTECHE

SCIENTIFIC BACKGROUND

More than 30 incurable neurodegenerative diseases are associated with genetic mutations characterised by expansions of short nucleotide repeats. Their common pathological development takes place via a phase transition of the primary RNA transcripts from a usual liquid state into a state exhibiting several congested clusters known as aberrant nuclear foci (also nuclear-specles, RNA granules, etc.), whose formation generally has a negative impact on cellular functions. The interaction of foci with RNA-binding proteins results in the inhibition of natural function of those proteins and consequently leads to fatal errors in the subsequent translation process. Recent studies revealed that the formation of congested foci can take place not only within the biological cells, but also in in-vitro systems. This opens up a possibility to investigate the associated phase transition and phase separation phenomena from a new perspective, i.e. by the methods that are conventionally used to study such phenomena in the field of physics.

Molecular mechanisms that are governing the formation of foci and their sensitivity to the sequence length are at present still not well resolved, however, it was recently suggested that unconventional RNA folding and base-pairing mechanisms, in particular G-quadruplex formation, might play a promotive role. Therefore, the formation of DNA/RNA quadruplex structures might in general be an important intermediate step in the process of various intracellular modifications associated with repeat expansions. The crucial open problems are:

· How the length of repetitive sequences affects their assembly into G-quadruplex structures and other higher order assemblies like G-wires, etc.,

· Why, how and at which conditions those self-assembled structures stimulate phase transition and phase-separation into the congested RNA foci,

· What are the physical nature and physical properties of thermodynamic phase forming the foci

· What are the possibilities for regulation of formation and disintegration of the foci with some suitable external additives or stimuli.

PROJECT OBJECTIVES

For repetitive nucleotide sequences of different lengths we will resolve a phase diagram of their thermodynamic phases and determine fundamental physical properties of those phases. As we expect that the chromonic liquid crystalline phases will occur, the primary properties of interest will be the liquid crystalline orientational order, the periodicity of chiral modulation, the dielectric and magnetic anisotropy, the visco-elastic coefficients, etc. We will also examine a possibility to tune the critical concentration region of different phases by some selected additives, such as monovalent and divalent cations (e.g. Na⁺, K⁺, Mg⁺⁺), crowding agents (e.g. PEG), antisense sequences, G-quadruplex binding ligands (e.g. porphyrins), and certain proteins with a known tendency to bind to the RNA foci (e.g. SF2, hnRNP-H, HuR,). The project is aimed at the following main objectives:

· Structural characterization of self-assembled higher-order structures (G-quadruplex, G-wires, etc.) formed by nucleotide repeat expansions of different lengths,

· Systematic mapping of various phases and phase transitions occurring in solutions of repeat expansions of different lengths - determination of the phase diagram,

· Elucidation of the effect of selected additives and/or external stimuli on the phase separation process and the formation of condensed foci.

Description of the phases of the project on the monthly (m) base from m1-m36:

Phase 1 - Formation of higher-order structures
Objective: Structural characterization of self-assembled higher-order structures (G-quadruplex, G-wires, etc.) formed by nucleotide repeat expansions of different lengths.
Tasks:	T1.1. - Preparation of materials and samples (m1-m18), T1.2. - Characterization of higher-order self-assembled structures formed in solution (m6-m24), T1.3 - Characterization of higher-order self-assembled structures observed in solidified films (m6-m24).
Expected results:	R1.1. - Solution and thin film type samples of repetitive DNA and RNA sequences of various lengths will be prepared. R1.2. - G-quadruplexes and other higher-order noncanonical structures in solution will be characterized by various experimental techniques. R1.3. - G-quadruplexes and other higher-order noncanonical structures formed on solid substrates by solution coating will be characterized by AFM imaging.
Deliverables:	Scientific papers, conference contributions, students’ seminars and diploma/master works, research news on local websites/magazines/radio/TV.

Phase 2 - Phases and phase transitions
Objective: Systematic mapping of various phases and phase transitions occurring in solutions of repeat expansions of different lengths - determination of the phase diagram.
Tasks:	T2.1 - Categorization of different thermodynamic phases and phase transitions (m12-m30), T2.2. - Determination of physical properties of different phases (m12-m30), T2.3. - Theoretical modeling of phase transitions (m1-m18).
Expected results:	R2.1. - Phase diagram of different phases will be determined for some selected repetitive sequences. R2.2. - Fundamental physical properties of the N* CLC phase will be resolved for some selected repetitive sequences. R2.3. – Theoretical model of the I-N* phase transition as a function of the length of a repetitive sequence will be constructed.
Deliverables:	Scientific papers, conference contributions, students’ seminars and diploma/master works, research news on local websites/magazines/radio/TV.

Phase 3 - Effects of additives and external stimuli
Objective: Elucidation of the effects of selected additives and/or external stimuli on the phase separation process and the formation of condensed foci.
Tasks:	T3.1. - Investigation of effect of (bio)chemical additives on transition to congested phases (m18-m36), T3.2. - Investigation of effect of external stimuli on transition to congested phases (m18-m36), T3.3. Theoretical modelling of the interaction of different phases with additives and external stimuli (m18-m36).
Expected results:	R3.1. - The effects of some selected additives on the I-N* phase transition will be characterized. R3.2. - The effects of external magnetic field and optical irradiation on the I-N* phase transition will be characterized. R3.3. - Theoretical model of effects of some selected additives/stimuli on the I-N* phase transition will be constructed.
Deliverables:	Scientific papers, conference contributions, students’ seminars and diploma/master works, research news on local websites/magazines/radio/TV.

INTERIM REPORT (May 2020)

Most of the research in the initial period of the project focused on research of the mechanisms of folding and aggregation processes in relatively short guanosine-rich DNA oligonucleotides. These are investigations that we planned mainly in the first work package of the project (DS1 - Formation of higher order structures). In our work we cooperated especially with the research group of prof. S. Masiero from the University of Bologna in Italy. Part of the activities also took place in cooperation with the Faculty of Science and Mathematics of the University of Maribor.

OBJECTIVE 1: Structural characterization of spontaneously formed highly-organized structures (Gquadruplexes, G-wires, etc.) based on repetitive nucleotide sequences of different lengths.

To achieve this goal, we performed an extensive analysis of the folding processes of guanosine-rich DNA sequences into G-quadruplex structures in solutions and in solidified films. Experiments in solutions are performed under conditions similar to (or corresponding to) physiological conditions. Using dynamic light scattering (DLS) technique in combination with atomic force microscopy (AFM), we investigated the mechanisms of folding into G-quadruplexes and their aggregation into G-wires of the sequences (G4C2)xn for n = 1, 2 and 4 [1, 2.3]. We also analyzed some other guanosine-rich DNA sequences of similar length [4-6] and human papillomavirus sequences [7], with NMR spectroscopy accounting for the majority of this research. In addition, we also investigated the interaction properties between different guanosine derivatives and derivatives of other nucleobases. This research is focused on the issue of base-pairing and base assembly in two-dimensional thin layers on the water surface [8]. We also studied spontaneously-formed highly-organized structures in solidified films transferred from the water surface to solid substrates [9,10].

OBJECTIVE 2: Systematic determination of different phases and phase transitions in solutions of repetitive nucleotide sequences of different lengths - determination of the phase diagram.

We also began research into the specific folding mechanisms of long strands of DNA with embedded (G4C2)x48 sequences. Atomic force microscopy (AFM) was used to analyze the aggregation properties of such DNA plasmids compared to analog plasmids with the same random sequence embedded. We also studied denaturation and renaturation mechanisms in the mentioned systems [11, 12]. Further research in this area is ongoing. We are also preparing the first scientific article on this topic.

OBJECTIVE 3: Determination of the effect of various additives and external stimuli on the phase separation process and the formation of condensed droplets.

For the realization of the above stated goal, it is first necessary to complete the first two work packages of the project, so the implementation of activities related to it is planned in the last year of project implementation. Among the various external stimuli, we will also be interested in the influence of UV light on the phase separation processes. For this purpose, we have recently developed a specially designed illumination system with two types of LED

REFERENCES

1. Y. Shin and C. P. Brangwynne, Liquid phase condensation in cell physiology and disease, Science 357, 1253 (2017).

2. A. J. Hannan, Tandem repeats mediating genetic plasticity in health and disease, Nat. Rev. Genet., Feb. 5 (2018).

3. A. R. La Spada and J. P. Taylor, Repeat expansion diseases: progress and puzzles in disease pathogenesis, Nat. Rev. Genet. 11, 247 (2010).