臺灣博碩士論文加值系統

　　多胺類是細胞中相當重要且無所不在的物質，且以相當高的含量存在細胞核裡。之前研究就已經發現多胺類可以用來安定核酸結構，不過直至目前為止，多胺類在雙螺旋的研究上並不是十分透徹。為了瞭解細胞內多胺類對自然界這些雙螺旋的作用，所以本諞研究設計了富含A-T鹼其配對B-form的雙螺旋；包括完美雙螺旋、非正常鹼基配對（A:G）雙螺旋、髮夾式雙螺旋和buldge雙螺旋。希望藉由物理方法（紫外線光譜，圓二色光譜和生物感應器）的測定，並透過進一步的動力學和熱力學計算分析，更清楚了解與證明多胺類在核酸結構多樣化的過程中可能扮演的角色。在熱力學實驗中我們發現多胺類比無機鹽類更能提高雙螺旋穩定性；其中spermine對於所有雙螺旋是沒有選擇性的，實驗結果顯示，在spermine影響下，所有雙螺旋的Tm會上升到最高值。尤其以spermine對於非正常雙螺旋（Ml、Bl和B2）具有相當好的修飾穩定作用。另外spermine會明顯增加非自形成螺旋的熱力學常數，但對髮夾式雙螺旋的熱力學常數則沒有影響。接下來的圖二色光譜實驗中，多胺類狀況下的雙螺旋光譜曲線特徵沒有太大改變。結果證明了不管是spermidine或spermine對於各式雙股螺旋只增加核酸穩定性但不會改變其結構。最後在動力學實驗中發現，鹽類和多胺類的存在下，雙螺旋（P3、M1和B2）的結合速率常數（ka）都在6~8×105，DNA結合的速度相當快速，不過在解離的部份上，spermidine狀況下的M1和B2之比在MgCl2狀況之解離速率常數（kd）下降2~3倍，甚至與P2的kd值並無差別。所以spermidine可以使非正常DNA hybridization更緊密。另外spermine比spermidine更能大大降低所有的雙螺旋解離速率常數到達10-4。尤其對於完美雙螺旋的kd也能夠發揮作用。這也與之前的熔點溫度結果相互呼應。細胞的核酸儲在細胞核中，為了不同生化代謝目的，其結構是以相當多樣化的型態來變化，本諞論文我們從物性上了解多胺類對各類核酸雙螺旋結構的穩定是不分彼此的，而間接印證在細胞核中的高多胺類確實對核酸結構具有相同穩定的功用。

　　Concentrations to polyamines are in millimolar and can be as high as 5mM in the eukaryotes nucleus. Due to their chemical structure, it was suggested that the polyamine can stabilize the nucleic acid secondary structures, such as duplex and triplex. The report presents a systematic study to reveal the role of salt, spermidine, and spermine in various duplex formation with rich A-T base pairs. The melting temperatures (Tm) and structure were measured with a few straight rod type duplexes with complete complementary base pairs; containing a bulge G base, with an AG mismatched, and with a hairpin structures by CD and UV spectra in various solutions containing spermidine and spermine. The results showed that the influence of spermine to Tm's are not the same but with the following order: bulge and mismatched dupleses>straight duplexes>hairpin duplexes. On the other hand the stability of these duplexes are reverse true without polyamines. The CD data indicate that the polypositive charged polyamines may effectively neutralize the negative charges of oligonucleotides sugar-phosphate backbone and make the duplexes more behaved in solution but didn't cause the change of conformation of duplexes. Finally, the kinetic impact of duplexes formation by polyamine is current studied by surface plasmon resonance phenomenon This replrt presents a novel strategy for using surface plasmon resonance to detect the kinetic constant of the effect of polyamines, such as spermine and spermidine, on the formation of DNA duplexes with Adenine rich sequence. The synthetic oligonucleeotide duplexes contain either a bulged base (B1), or a mismatch base pair (M1) or without lesions (P1). Results showed that the association rate constants are in the range of 106M-1S-1 in all duplexes under various conditions. This indicates that the formation of duplex is instantaneous. On the other hand, the dissociation rate constants have the following order: M1 and B1>P1 with cations presence; However, the difference of the dissociation constant of M1,B1 and P1 are dismissed. This indicated that polyamine can compenstae the energy discrepancy caused by lesions (e.g. bulged loop or mismatched base pairs) in the duplex formation. Our study may provide a basis for explaining the DNA polymorphism phenomenon, since the spermine is ubiquitous in living cell.