Probe The Formation Of Quadruplex Of Telomere DNA And Study On Its Kinetic Properties

DNA and RNA may fold into sencondary structure via intramolecular interaction. The formation and stability of such structure have great biological significance. They can serve as therapeutic targets for cancers and other diseases due to their potential biological function. Human telomere consists of tandem repeats of TTAGGG and CCCTAA . It is well known that the G strand can stack via Hoogsteen hydrogen bonding into G-quadruplex and the C strand can also fold into i-motif by intercalated C-C⁺ base pairing. Once G-quadruplex is formed, telomerase activity may be inhibited because telomerase can not recognize it as substrate. Therefore, screening of molecules targeting and stabilizing such structure springs up as new hope in the field of cancer therapy.DNA chip technology provides a powerful tool for screening molecules. To seek for molecules targeting secondary structure such as G-quadruplex, however, an important issue we should address is the formation and stability of the secondary structure formed by the immobilized DNA.In our work, we qualitatively and quantitatively studied the immobilized telomere DNA using Biacore technology. The following are the main results.I) Probing the formation of tetraplex by single strand binding protein (SSB)1. Using UV irradiation, the quadruplex formed by (TTAGGG)4 was crosskinked and purified. Gel mobility shift assay demonstrated that SSB did not bind to the crosslinked quadruplex, but only to the single-stranded form. Based on this property, SSB can be used as an universal probe to detect the formation of secondary structure.2. (TTAGGG)4 showed a faster migration than both single strand and duplex DNA with the same length in K⁺, indicating that it formed secondary structure. The results of the melting experiments showed typical transition curves in Na⁺ and K⁺ but not in Li⁺. The tansition temperature was higher in K⁺ than in Na⁺. These results demonstrated that K⁺ can promote G strand to form secondary structure more effectively than Na⁺.3. The binding of SSB to immobilized DNA was analyzed by Biacore. Comparing with Li⁺, K⁺can strongly inhibit the binding of SSB to (TTAGGG)₄, whileit can slightly increase the binding of SSB to random sequence that does not form structure. Our experiment proved that the inhibition is caused by the formation of quadruplex by G strand.4. The formation of i-motif by (CCCTAA)4 was followed by absorbance at 260nm and gel filtration chromatography. Decreasing in pHresulted in an increasing in melting tempreture and decreasing in the retention time in chromatography.5. The bingding of SSB to the immobilized (CCCTAA)4 was inhibited by proton. Taking into consideration that proton can slightly enhance the bingding of SSB to random sequence. The inhibition can only be explained by i-motif formation.From those results, immobilized DNA can form secondary structure just as they did in solution. SSB can probe the formation of such structures under various conditions.II Kinetic properties of tetraplex formation1. When hybridizing G strand immobilized on the chip with its complementary strand, the binding is inhibited by Na+ and K+ but not Li+, while the hybridization of random sequence as a control is not aflfected by ion. Therefore, the inhibition of the hybridization of G strand by K+ is caused by the formation of quadruplex. This result demonstrated that like single srand binding protein, complementary strand hybridization can also probe the formation of quadruplex.2. Immobilized DNA can adopt two conformations known as folded form and unfolded form and its complementary sequence.can only bind to the unfolded form. By coupling the intramolecular reaction with intermolecular hybridization, we developed a method measure the intramolecular folding and unfolding rate constants, and the intermolecular association and dissociation rate constants as well.3. To prove the theory, we obtained kinetics curves by hybridization C strand with the immobilized G strand. The folding and unfolding rate constant of quadruplex formation and the association and dissociation rate constant of duplex formation were simultaneously extracted by fitting the data with the method. The sensorgrams both in Na+ and in k+ can be fitted well by coupled model but not by simple model. However, the sensorgrams obtained in Li+ can be fitted by both models. As a control, the sensorgrams of random sequence hybridization obtained in each cation can be fitted by both models. These results proved that coupled model can reflect the real reactionof the formation of secondary structure, considering that Na+ and K+ but not Li+ can promote the formation of quadruplex.4. To study the kinetic property of the quadruplex formation by different sequences, we selected four G-rich sequences out of several sequcences by electrophoresis. The unique band of each sequence when electrophoresis even at high concentration indicated that they have purely formed quadruplex , as is supported by melting experiment. The sensorgrams of each sequence obtained by hybridization at the same condition were fitted well by the coupled model, also indicating that they formed quadruplex. The kinetic parameters were extracted by fitting. It turned out that small changes of loop and base composition may have profound effects on the formation of quadruplex and its stability. With the increasing of the loop length and decreasing of G base, both of the folding rate constant decreased while the unfolding rate constant changed a little. Therefore, the decreased stability of the quadruplex by the change of loop and base composition mainly resulted from the increasing difficulty of folding. The data obtained by changing the approach of DNA immobilization suggested that steric effect and decreasing flexibility may also led to the increasing difficulty of folding.In summary, our method can not only probe the formation of the secondary structure of immobilized DNA, but also extract the four kinetics parameter simutaneously. The measurement of the folding and unfolding rate constant of different sequences demonstrated that loop and base composition and the immobilization approach can all have great effect on the quadruplex formation and stability.