Projects / Programmes
Stability of novel AGCGA quadruplexes and their recognition by nanobodies
| Code |
Science |
Field |
Subfield |
| 1.04.02 |
Natural sciences and mathematics |
Chemistry |
Structural chemistry |
| Code |
Science |
Field |
| P003 |
Natural sciences and mathematics |
Chemistry |
| Code |
Science |
Field |
| 1.04 |
Natural Sciences |
Chemical sciences |
quadruplex, nanobody, AGCGA repeat, structure, folding, binding, energetics
Researchers (7)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
36924 |
PhD San Hadži |
Chemistry |
Researcher |
2019 - 2022 |
92 |
| 2. |
54005 |
Mojca Hunski |
Chemistry |
Researcher |
2020 - 2021 |
4 |
| 3. |
15669 |
PhD Jurij Lah |
Chemistry |
Head |
2019 - 2022 |
337 |
| 4. |
03422 |
PhD Brigita Lenarčič |
Biochemistry and molecular biology |
Researcher |
2019 - 2021 |
338 |
| 5. |
23575 |
PhD Miha Pavšič |
Biochemistry and molecular biology |
Researcher |
2019 - 2022 |
204 |
| 6. |
15293 |
PhD Črtomir Podlipnik |
Chemistry |
Researcher |
2019 - 2022 |
219 |
| 7. |
53449 |
PhD Uroš Zavrtanik |
Chemistry |
Junior researcher |
2019 - 2022 |
30 |
Organisations (1)
Abstract
In 1953 Watson and Crick presented their iconic structure of double stranded DNA. Since then, this structure has become firmly engraved in the cultural memory of mankind. Yet, DNA is also capable of adopting other structures that are appear only in specific contexts. For example, widely recognized are the so-called G-quadruplexes (G4) formed from G-rich sequences and i-motifs which form from the complementary strand. Although these structures have been extensively studied their role for the gene regulation and their links with diseases such as cancer, Alzheimer and diabetes is still a matter of extensive research. Traditionally noncanonical DNA structures were studied in vitro and their existence in the cells was matter of debate. This question has been settled by landmark studies which relied on detection of G4 and i-motifs using specific antibodies developed using the synthetic antibody libraries.
Recently, a new fold on noncanonical DNA structure termed AGCGA-quadruplex has been discovered. At the sequence level these structures adhere to a repeat of the 5'-AGCGA-3' motif. At the structural level these quadruplexes are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets. In stark contrast to G-quadruplexes these structures are not stabilized by specific cation binding and are thus insensitive to the nature of cation. The small-scale bioinformatic study has identified AGCGA-quadruplex sequence motifs in over 40 human genes related to the regulation of basic cellular processes, neurological disorders, cancer and abnormalities in bone and cartilage development, while mutation of motifs has been linked to autism.
The main goal of the proposed project is to establish whether AGCGA-quadruplexes are formed in vivo. This will require development of detection tools, specific ligands that will recognize AGCGA-quadruplex in the cellular, native context. We plan to develop of a panel of nanobodies (single-chain camelid antibodies) specifically recognizing AGCGA-quadruplex. Nanobody-AGCGA-quadruplex interactions will be studied in terms of affinity, specificity, selectivity and structural aspects of recognition, resulting in a set of well-characterized reagents for the AGCGA-quadruplex detection. Ultimately, these nanobodies will be used for detection of AGCGA-quadruplex in cells by immunoimaging techniques. In parallel we also propose to study stability of the AGCGA-quadruplex in vitro using biophysical techniques to identify how solution conditions (cation concentration, water activity, molecular crowding) affect its stability. This will corroborate findings from the in vivo studies and provides a complementary approach to assess whether AGCGA-quadruplex are stable in vivo. Establishing the potential existence of a novel type of noncanonical DNA structure will give a strong impetus in the field of noncanonical DNA biology, while the developed of AGCGA-quadruplex specific antibodies will provide tools for the scientific community promoting further research.
Significance for science
Other studies which focused on detecting G4 and i-motif structures used specific Fab antibodies. This type of antibody construct significantly differs from nanobodies which, to our knowledge, have not been used to target any non-canonical DNA structures so far. An important advantage of nanobodies is that they may be used as crystallization chaperones with high success (there are over twice as much nanobody-target complexes in PDB database compared to Fab complexes). Compared to other studies developing antibodies targeting non-canonical DNA structures we will give a strong emphasis on the characterization of nanobody-target recognition at molecular level (selectivity, precise determination of affinity, cross-reactivity and, crucially, structural aspects of quadruplex recognition). In this light, we want to deliver a novel, powerful and well-characterized tool to the scientific community. Furthermore, the complementary picture regarding AGCGA stability will be provided by thermodynamic analysis (Task 3), which will give deeper insights into the driving forces that stabilize these structures.
The expected results of the project, the assessment of AGCGA-quadruplex stability in vivo, will be an important and unique result in the field of non-canonical DNA biology. If the presence of novel structure is established in vivo, this will stimulate further research of the physiological relevance of these structures. Furthermore, the proposed development of toughly characterized nanobodies targeting AGCGA-quadruplexes will provide a powerful tool for the scientific community. It should be also mentioned that so far, no structural data is available on antibody-noncanonical DNA and protein recognition of non-canonical DNA structures is extremely scarce and limited only to peptide models. A possible outcome of this project (nanobody-quadruplex crystal structures) will fill this gap and shed light on how can proteins recognize non-canonical DNA structures.
Significance for the country
Other studies which focused on detecting G4 and i-motif structures used specific Fab antibodies. This type of antibody construct significantly differs from nanobodies which, to our knowledge, have not been used to target any non-canonical DNA structures so far. An important advantage of nanobodies is that they may be used as crystallization chaperones with high success (there are over twice as much nanobody-target complexes in PDB database compared to Fab complexes). Compared to other studies developing antibodies targeting non-canonical DNA structures we will give a strong emphasis on the characterization of nanobody-target recognition at molecular level (selectivity, precise determination of affinity, cross-reactivity and, crucially, structural aspects of quadruplex recognition). In this light, we want to deliver a novel, powerful and well-characterized tool to the scientific community. Furthermore, the complementary picture regarding AGCGA stability will be provided by thermodynamic analysis (Task 3), which will give deeper insights into the driving forces that stabilize these structures.
The expected results of the project, the assessment of AGCGA-quadruplex stability in vivo, will be an important and unique result in the field of non-canonical DNA biology. If the presence of novel structure is established in vivo, this will stimulate further research of the physiological relevance of these structures. Furthermore, the proposed development of toughly characterized nanobodies targeting AGCGA-quadruplexes will provide a powerful tool for the scientific community. It should be also mentioned that so far, no structural data is available on antibody-noncanonical DNA and protein recognition of non-canonical DNA structures is extremely scarce and limited only to peptide models. A possible outcome of this project (nanobody-quadruplex crystal structures) will fill this gap and shed light on how can proteins recognize non-canonical DNA structures.
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