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Young Researchers and Professionals
| Project leader |
Vuletić Tomislav |
| Administering organization: |
Institut za fiziku, p.p. 304, 10000 Zagreb, www.ifs.hr,
represented by Director, Dr. sc. Milorad Milun, milun@ifs.hr
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| Partner Institution/Company: |
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| Grant type: |
3A |
| Project title: |
Protein Assisted DNA Monolayer Assembly |
| Project summary: |
Rapid detection of specific DNA sequences, so-called DNA-sensing, has important technological applications, but also raises several fundamental questions in the field of biophysics. Studies of DNA sensing are very widespread and intense, and instead of performing the tests by time consuming methods in solutions, today’s methods concentrate on immobilization of DNA probes on substrates – i.e. on massive parallel processing of target molecules on DNA sensors/chips. However, the feasibility and speed of processes occurring on these sensors are highly dependent on the accessibility of the genetic code, which is itself determined by the conformation of the DNA molecule on the surface. We here propose to develop a novel array of fully extended, hence fully accessible, DNA molecules which will enable us to overcome some of the important limitations of previously designed sensors. Developing such an original approach, is a feat in itself, but is nevertheless necessary in order to give us a competitive edge. Successful accomplishment will provide us with a solid groundwork for studies of specific DNA interactions in two-dimensional geometries as well as direct us toward exploring possibilities for technological innovations.
We intend to utilize in a rather original manner the natural functional structure of DNA and RecA protein complexes – nucleoprotein filaments. We propose that it is possible to attach DNA molecules to the substrate in a uniform and organized way, while forming nucleoprotein filaments which have a large persistence length. Thus we expect to obtain a self-assembled monolayer of densely packed rod-like nucleoprotein filaments in “brush”-like conformation. Both, single or double stranded DNA probes, with much larger range of lengths will be used as probes, thus providing a single flexible approach to DNA sensor preparation.
The research of the formation of such a layer will also give insight into fundamental concepts of charged polymers like coil-globule and/or brush-mushroom transition or the persistence length in restricted 2D geometry. This work is based on the team’s complementary experience with DNA/RecA complexes preparation, chemistry of DNA/substrate interactions, impedance spectroscopy and nonlinear laser optics for detection, as well as on the expertise in modeling of soft-matter interfaces.
An essential step of the current project is the construction of a 2-photon absorption fluorescence correlation spectroscopy instrument that can detect the anticipated low DNA-target/probe concentration-changes in a most precise manner. Beyond the direct scope of the current proposal, but as its consequence, the scientific community in Croatia will have direct access to this advanced technique.
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| Hrvatski sažetak: |
Potreba za brzim sekvenciranjem DNK molekula, u kliničkoj i znanstvenoj primjeni, otvara niz novih, fundamentalnih pitanja u području biofizike. Umjesto vremenski zahtjvenog rada s otopinama, današnje se metode baziraju na stvaranju DNK senzora/čipova. Oni nastaju učvršćivanjem DNK proba na razne podloge (substrate), i na takav način služe kao detektori za određenu vrstu molekula (ciljane molekule) koje prepoznaju DNK probu. Ostvarivost i brzina procesa koji se odvijaju na senzorima ovise o dostupnosti genetičkog koda koji je određen načinom slaganja DNK molekule na površinu podloge. U ovom projektu, izlažemo ideju o stvaranju DNK senzora koji bi se sastojao od podloge na koju je smještena potpuno izdužena DNK molekula - proba, koja time postaje potpuno dostupna za interakciju sa ciljanom DNK. Time bi se zaobišla neka od važnih ograničenja do sada poznatih koncepata DNK senzora.
Naša je ideja da na jedan potpuno originalan način iskoristimo prirodnu funkcionalnu strukturu koju stvara RecA protein polimerizacijom oko DNK molekule – strukturu koja se naziva nukleoproteinski lanac. Predlažemo da je moguće učvrstiti DNK molekulu na podlogu na uniforman i organiziran način kao posljedicu relativne čvrstoće i rigidnosti tih nukleoproteinskih lanaca u odnosu na samu DNK. Kao krajnji rezultat očekujemo stvaranje samoorganizirajućeg monosloja gusto pakiranih, štapićastih nukleoproteinskih lanaca postavljenih na podlogu tako da nalikuju na gustu «četku»! U tim slojevima koristiti ćemo kao probe jedno- i dvolančane DNK, različitih duljina, istražujući na takav način najbolju kombinaciju za stvaranje prethodno opisanog DNK senzora.
Istraživanja procesa samo-organizacije gusto pakiranog mono-sloja omogućila bi nove fundamentalne spoznaje u srodnim sistemima, npr. nabijenih polimera - polielektrolita i njihovih prijelaza iz stanja savijenog lanca u kompaktno namotano stanje, prijelaza polielektrolita na površinama iz izduženog oblika u savijeni oblik, spušten na površinu podloge, ili na istraživanja utjecaja na duljine tvrdokornosti polielektrolita zbog njihove organizacije u samo dvije dimenzije.
Jedan od ključnih koraka u predloženom projektu je izgradnja optičkog postava za fluorescencijsku korelacijsku spektroskopiju, ali baziranu na 2-fotonskoj apsorpciji, pomoću kojeg bi se moglo na vrlo precizan način mjeriti promjene u koncentraciji probnih i ciljanih DNK molekula. Ovakav visoko sofisticirani instrument, proizašao bi kao direktna posljedica predloženog projekta, međutim njegovo korištenje ne bi bilo ograničeno samo na suradnike na projektu, već bi bio ponuđen na korištenje cjelokupnoj hrvatskoj znanstvenoj zajednici. |
| Amount requested from UKF: |
50.000 € |
| Amount of matching funding: |
30.000 € |
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