Multiple Labeling of DNA and RNA with NIR fluorophores by means of (bio)orthogonal approaches  részletek

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Projekt adatai

  
azonosító
110214
típus NN
Vezető kutató Kele Péter
magyar cím Multiple Labeling of DNA and RNA with NIR fluorophores by means of (bio)orthogonal approaches
Angol cím Multiple Labeling of DNA and RNA with NIR fluorophores by means of (bio)orthogonal approaches
magyar kulcsszavak NIR, bioorthogonal, oligonucleotides, labeling
angol kulcsszavak NIR, bioorthogonal, oligonucleotides, labeling
megadott besorolás
Szerves-, biomolekuláris- és gyógyszerkémia (Műszaki és Természettudományok Kollégiuma)100 %
Ortelius tudományág: Intelligens anyagok
zsűri Kémia 2
Kutatóhely Szerves Kémiai Intézet (HUN-REN Természettudományi Kutatóközpont)
résztvevők Cserép Balázs Gergely
Herner András
projekt kezdete 2013-07-01
projekt vége 2016-06-30
aktuális összeg (MFt) 22.653
FTE (kutatóév egyenérték) 5.41
állapot lezárult projekt
magyar összefoglaló
A kutatás összefoglalója, célkitűzései szakemberek számára
Itt írja le a kutatás fő célkitűzéseit a témában jártas szakember számára.
In DNA based molecular diagnostic tools and probes for cellular imaging the attachment of fluorescent labels to DNA is of paramount importance. Particularly, multiple labeling tools have turned out to be very useful in the fabrication of DNA-based optically functionalized architectures. Such oligonucleotides are mostly labeled post-synthetically as the applicable labels are limited to those that can withstand the harsh conditions applied during DNA synthesis. The ideal solution would be the use of near infra-red (NIR) labels to eliminate the aforementioned problems. Herein we propose the development of a generally applicable technique for the implementation of nucleotides into short and longer sequences (DNA or RNA) that enable post-synthetic incorporation of NIR fluorescent tags. To facilitate high yielding and efficient multiple labeling tasks under in vivo conditions, a special set of “click”-type reactions, namely bioorthogonal reactions are to be used. To accomplish the aimed work a series of phosphoramidite derivatives furnished with appropriate bioorthogonal functions are proposed that shall be incorporated into short oligos by automated DNA synthesis and in longer oligos by means of DNA polymerases. Parallel to this, a library of near-IR emitting, turn-on type bioorthogonalized fluorophores with large Stokes-shifts will be constructed. NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.

Mi a kutatás alapkérdése?
Ebben a részben írja le röviden, hogy mi a kutatás segítségével megválaszolni kívánt probléma, mi a kutatás kiinduló hipotézise, milyen kérdéseket válaszolnak meg a kísérletek.
The chemistry for attachment of labels onto biomolecules is of utmost importance in many applications of biomedical research. New strategies for fluorescent probe design and imaging require the combination of intelligent organic synthesis, chemical biology methods, theoretical calculations and profound photophysical understanding of fluorophore interactions (excitons, excimers, exciplexes, energy and electron transfer).
In many cases the sensitivity of fluorescence detection approaches that of radiolabeling with the advantage of higher stability and without the health damaging effects of radioactive radiation. Moreover, multicolor fluorescence detection allows the detection and resolution of multiple targets using fluorescent labels that can be spectrally resolved. The ability to detect and analyze two or more labeled targets in the same sample eliminates sample-to-sample variation and is both time- and cost-effective.

Mi a kutatás jelentősége?
Röviden írja le, milyen új perspektívát nyitnak az alapkutatásban az elért eredmények, milyen társadalmi hasznosíthatóságnak teremtik meg a tudományos alapját. Mutassa be, hogy a megpályázott kutatási területen lévő hazai és a nemzetközi versenytársaihoz képest melyek az egyediségei és erősségei a pályázatának!
NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.

A kutatás összefoglalója, célkitűzései laikusok számára
Ebben a fejezetben írja le a kutatás fő célkitűzéseit alapműveltséggel rendelkező laikusok számára. Ez az összefoglaló a döntéshozók, a média, illetve az érdeklődők tájékoztatása szempontjából különösen fontos az NKFI Hivatal számára.
In DNA based molecular diagnostic tools and probes for cellular imaging the attachment of fluorescent labels to DNA is of paramount importance. Particularly, multiple labeling tools have turned out to be very useful in the fabrication of DNA-based optically functionalized architectures. Such oligonucleotides are mostly labeled post-synthetically as the applicable labels are limited to those that can withstand the harsh conditions applied during DNA synthesis. The ideal solution would be the use of near infra-red (NIR) labels to eliminate the aforementioned problems. Herein we propose the development of a generally applicable technique for the implementation of nucleotides into short and longer sequences (DNA or RNA) that enable post-synthetic incorporation of NIR fluorescent tags. To facilitate high yielding and efficient multiple labeling tasks under in vivo conditions, a special set of “click”-type reactions, namely bioorthogonal reactions are to be used. To accomplish the aimed work a series of phosphoramidite derivatives furnished with appropriate bioorthogonal functions are proposed that shall be incorporated into short oligos by automated DNA synthesis and in longer oligos by means of DNA polymerases. Parallel to this, a library of near-IR emitting, turn-on type bioorthogonalized fluorophores with large Stokes-shifts will be constructed. NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.
angol összefoglaló
Summary of the research and its aims for experts
Describe the major aims of the research for experts.
In DNA based molecular diagnostic tools and probes for cellular imaging the attachment of fluorescent labels to DNA is of paramount importance. Particularly, multiple labeling tools have turned out to be very useful in the fabrication of DNA-based optically functionalized architectures. Such oligonucleotides are mostly labeled post-synthetically as the applicable labels are limited to those that can withstand the harsh conditions applied during DNA synthesis. The ideal solution would be the use of near infra-red (NIR) labels to eliminate the aforementioned problems. Herein we propose the development of a generally applicable technique for the implementation of nucleotides into short and longer sequences (DNA or RNA) that enable post-synthetic incorporation of NIR fluorescent tags. To facilitate high yielding and efficient multiple labeling tasks under in vivo conditions, a special set of “click”-type reactions, namely bioorthogonal reactions are to be used. To accomplish the aimed work a series of phosphoramidite derivatives furnished with appropriate bioorthogonal functions are proposed that shall be incorporated into short oligos by automated DNA synthesis and in longer oligos by means of DNA polymerases. Parallel to this, a library of near-IR emitting, turn-on type bioorthogonalized fluorophores with large Stokes-shifts will be constructed. NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.

What is the major research question?
Describe here briefly the problem to be solved by the research, the starting hypothesis, and the questions addressed by the experiments.
The chemistry for attachment of labels onto biomolecules is of utmost importance in many applications of biomedical research. New strategies for fluorescent probe design and imaging require the combination of intelligent organic synthesis, chemical biology methods, theoretical calculations and profound photophysical understanding of fluorophore interactions (excitons, excimers, exciplexes, energy and electron transfer).
In many cases the sensitivity of fluorescence detection approaches that of radiolabeling with the advantage of higher stability and without the health damaging effects of radioactive radiation. Moreover, multicolor fluorescence detection allows the detection and resolution of multiple targets using fluorescent labels that can be spectrally resolved. The ability to detect and analyze two or more labeled targets in the same sample eliminates sample-to-sample variation and is both time- and cost-effective.

What is the significance of the research?
Describe the new perspectives opened by the results achieved, including the scientific basics of potential societal applications. Please describe the unique strengths of your proposal in comparison to your domestic and international competitors in the given field.
NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.

Summary and aims of the research for the public
Describe here the major aims of the research for an audience with average background information. This summary is especially important for NRDI Office in order to inform decision-makers, media, and others.
In DNA based molecular diagnostic tools and probes for cellular imaging the attachment of fluorescent labels to DNA is of paramount importance. Particularly, multiple labeling tools have turned out to be very useful in the fabrication of DNA-based optically functionalized architectures. Such oligonucleotides are mostly labeled post-synthetically as the applicable labels are limited to those that can withstand the harsh conditions applied during DNA synthesis. The ideal solution would be the use of near infra-red (NIR) labels to eliminate the aforementioned problems. Herein we propose the development of a generally applicable technique for the implementation of nucleotides into short and longer sequences (DNA or RNA) that enable post-synthetic incorporation of NIR fluorescent tags. To facilitate high yielding and efficient multiple labeling tasks under in vivo conditions, a special set of “click”-type reactions, namely bioorthogonal reactions are to be used. To accomplish the aimed work a series of phosphoramidite derivatives furnished with appropriate bioorthogonal functions are proposed that shall be incorporated into short oligos by automated DNA synthesis and in longer oligos by means of DNA polymerases. Parallel to this, a library of near-IR emitting, turn-on type bioorthogonalized fluorophores with large Stokes-shifts will be constructed. NIR (multiply) tagged oligos like molecular beacons or siRNAs will enable autofluorescence free imaging of live tissues and their use would open up new ways in autofluorescence free, deep-tissue imaging of biological samples in the future and can offer new means for e.g. the early detection of cancer.




 

Zárójelentés

  
kutatási eredmények (magyarul)
A projekt keretében célul tűztük ki bioortogonális reagensek, valamint bioortogonalizált fluoreszcens jelzővegyületek fejlesztését és szintézisét, melyek DNS molekulák sejten belüli vizsgálatát teszik lehetővé. Ilyen vegyületek lehetővé teszik olyan eljárások kifejlesztését, melyek segítségével szelektíven manipulálható nukleotid építőelemek építhetők be hosszabb-rövidebb nukleinsavakba akár szilárdfázisú szintézissel, akár PCR-technológia segítségével, enzimatikusan. A támogatási időszak alatt sikeresen állítottunk elő bioortogonalizált foszforamidit-származékokat, melyek lehetővé tették e nukleotidok szintetikus DNS-be építését. Vizsgáltuk továbbá az így módosított szintetikus nukleinsavak jelölhetőségét is fluoreszcens jelzővegyületekkel. Ezen kívül megfelelően bioortogonalizált nukleotid trifoszfát-származékokat is előállítottunk és vizsgáltuk ezek enzimatikus beépíthetőségét PCR technológia segítségével. E fejlesztésekkel párhuzamosan előállítottunk számos fluoreszcens és fluorogén jelzővegyületet, melyek a távoli vörös, közeli infravörös tartományban emittálnak és a megfelelően módosított DNS molekulák bioortgonális jelölésére alkalmasak. E fejlesztések nagyban hozzájárulnak a molekuláris biológiai folyamatok megértéséhez, vagy alkalmazhatók az orvosdiagnosztikai eljárásokban.
kutatási eredmények (angolul)
Within this project we aimed at developing biorthogonal reagents and bioorthogonalized fluorescent probes for cellular imaging of DNA. Such optically functionalized DNA architectures can be used for the better understanding of biomolecular processes or in diagnostic processes. We proposed the development of a generally applicable technique for the implementation of nucleotides into short and longer sequences (DNA or RNA) that enable post-synthetic incorporation of fluorescent / fluorogenic tags. Upon systematic examination and development of biorthogonal reagents we have synthesized phosphoramidite derivatives furnished with appropriate bioorthogonal functions that enabled incorporation of such building blocks into short oligos by automated DNA synthesis. Furthermore, we also investigated the possibility of incorporation of as-functionalized nucleotide triphosphates into longer oligos by means of DNA polymerases. Parallel to this work, we have accomplished the synthesis and study of a set of near-IR emitting or fluorogenic labels that offer post-synthetic optical manipulation od DNA (RNA) by means of biorthogonal transformations.
a zárójelentés teljes szövege https://www.otka-palyazat.hu/download.php?type=zarobeszamolo&projektid=110214
döntés eredménye
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Közleményjegyzék

  
Söveges, B.; Imre, T.; Szende, T.; Póti, Á. L.; Cserép, G. B.; Hegedűs, T.; Kele, P.; Németh, K.: Systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags, Org. Biomol. Chem. 2016, 14, 6071-6078, 2016
Eördögh, Á.; Steinmeyer, J.; Peewasan, K.; Schepers, U.; Wagenknecht, H-A.; Kele, P.: Polarity sensitive bioorthogonally applicable far-red emitting labels for postsynthetic nucleic acid labeling by copper-catalyzed and copper-free cycloaddition, Bioconjugate Chem. 2016, 27(2), 457-464, 2016
Demeter, O.; Fodor, E. A.; Kállay, M.; Mező, G.; Németh, K.; Szabó, P. T.; Kele, P.: A Double-Clicking Bis-Azide Fluorogenic Dye for Bioorthogonal Self-Labeling Peptide Tags, Chem. Eur. J. 2016, 22, 6382-6388, 2016
Knorr, G.; Kozma, E.; Herner, A.; Lemke, E. A.; Kele, P.: New, red-emitting tetrazine-phenoxazine fluorogenic labels for live-cell intracellular bioorthogonal labeling schemes, Chem. Eur. J. 2016, 22, 8972-8979, 2016
Cserép, G. B.; Herner, A.; Kele, P.: Bioorthogonal fluorescent labels: a review on combined forces, Methods Appl. Fluoresc. 2015, 3(4), 042001, 2015
Cserép, G. B.; Demeter, O.; Bätzner, E.; Kállay, M.; Wagenknecht, H-A.; Kele, P.: Synthesis and Evaluation of Nicotinic Acid Derived Tetrazines for Bioorthogonal Labeling, Synthesis 2015, doi: 10.1055/s-0034-1380721, 2015
Huber M C, Schreiber A, von Olshausen P, Varga BR, Kretz O, Joch B, Barnert S, Schubert R, Eimer S, Kele P, Schiller SM: Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments, NAT MATER 2015: (14) 125-132, 2015
Cserép, G. B.; Demeter, O.; Bätzner, E.; Kállay, M.; Wagenknecht, H-A.; Kele, P.: Synthesis and Evaluation of Nicotinic Acid Derived Tetrazines for Bioorthogonal Labeling, Synthesis 2015, doi: 10.1055/s-0034-1380721, 2015
Cserép Balázs Gergely, Baranyai Zsuzsa, Komáromy Dávid, Horváti Kata, Bősze Szilvia, Kele Péter: Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags, TETRAHEDRON 70: pp. 5961-5965., 2014
Herner A, Girona G E, Nikić I, Kállay M, Lemke E A, Kele P: New Generation of Bioorthogonally Applicable Fluorogenic Dyes with Visible Excitations and Large Stokes Shifts, BIOCONJUGATE CHEMISTRY 25:(7) pp. 1370-1374., 2014
Huber M C, Schreiber A, von Olshausen P, Varga BR, Kretz O, Joch B, Barnert S, Schubert R, Eimer S, Kele P, Schiller SM: Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments, NAT MATER 2015: (14) 125-132, 2015
Stubinitzky C, Cserep GB, Bätzner E, Kele P, Wagenknecht HA: 2’-Deoxyuridine Conjugated with a Reactive Monobenzocyclooctyne as a DNA Building Block for Copper-Free Click-type Postsynthetic Modification of DNA, CHEM COMMUN 50: 11218-11221, 2014
Cserép Balázs Gergely, Baranyai Zsuzsa, Komáromy Dávid, Horváti Kata, Bősze Szilvia, Kele Péter: Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags, TETRAHEDRON 70: pp. 5961-5965., 2014
Herner A, Girona G E, Nikić I, Kállay M, Lemke E A, Kele P: New Generation of Bioorthogonally Applicable Fluorogenic Dyes with Visible Excitations and Large Stokes Shifts, BIOCONJUGATE CHEMISTRY 25:(7) pp. 1370-1374., 2014
Stubinitzky C, Cserep GB, Bätzner E, Kele P, Wagenknecht HA: 2’-Deoxyuridine Conjugated with a Reactive Monobenzocyclooctyne as a DNA Building Block for Copper-Free Click-type Postsynthetic Modification of DNA, CHEMICAL COMMUNICATIONS Paper 10.1039/C4CC02855D., 2014
Cserép G B, Herner A, Wolfbeis O S, Kele P: Tyrosine specific sequential labeling of proteins, BIOORG MED CHEM LETT 23: 5776-5778, 2013




 

Projekt eseményei

  
2016-01-08 14:42:29
Résztvevők változása
2013-09-25 18:57:16
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