近年來，隨著生物技術的進步，核酸類似物例如：Locked nucleic acid(LNA)、Peptide nucleic acid(PNA)等成為新型生物技術應用於分子檢測的理想選擇。為了增加檢測的專一性與辨識性，我們實驗室發展一種新的電中性核酸類似物，phosphate-methylated DNA，並將其簡稱為nDNA。nDNA磷酸骨幹上不同核苷酸間的氧可以選擇性地經由甲基化(-CH3)，使帶負電的磷酸二酯鍵(O=P-O-，phosphodiester linkages)轉變成磷酸三酯鍵(O=P-O-CH3，methyl phosphotriester linkages)而成為電中性。此甲基化結果會使nDNA和其互補股核酸之間的靜電排斥力下降，而增加雙股的穩定性，但同時nDNA磷酸骨幹上的甲基也會在雙股螺旋形成時導致立體障礙的影響產生，並使結構不穩定。依此，就有機會藉由改變特定甲基化位置及不同數目，我們就能選擇與控制nDNA和互補股DNA雜交之性質，以為特定功能使用。 實驗結果方面，根據電泳我們可以分辨出不同數目nDNA的帶電量，並且證實nDNA能夠與一般DNA形成雜交，且circular dichroism spectroscopy(CD)的量測結果發現在波長275nm時出現一正峰，在245nm時出現負峰，指出nDNA和一般DNA雜交仍會形成B-form結構，值得注意的是我們藉由恆溫滴定微卡計(ITC)和melting temperature(Tm)的量測發現nDNA與一般DNA雜交，有可能會使雙股螺旋不穩定。而從∆G和Na+的斜率可以看到當雙股螺旋形成時，一般需要大量鹽離子以穩定結構，但若將其中一股換成nDNA能夠使Na+的吸收減少，同時我們也從熱力學參數探討無論是鈉離子或者是修飾甲基對於遮蔽磷酸骨幹上負電荷的影響，這一方面也使我們更加了解nDNA與其互補股之間的雜交機制。 最後，文獻指出辨識SNP特別是在GC-rich的序列是十分困難的，因為perfect match和mismatch之間的Tm值差異很小，所以我們選用了C型肝炎的其中一種分型(HCV-3b)當作目標物，此一序列為GC含量75%的序列，藉由改變nDNA修飾的位置與間隔不同鹼基數目修飾nDNA，發現間隔兩個鹼基設計nDNA或者是將nDNA設計在mismatch周圍，都能提高∆Tm，使得辨識能力上升，提供最佳化之設計的參考。 ;For the upsurging needs of disease detection and genetic analysis, the uses of synthetic DNA containing modified nucleotides such as LNA or PNA are becoming desired for novel biotech applications. To enhance the specificity of detections, we used newly developed DNA oligonucleotide (nDNA), which contains site-specific neutral internucleoside methyl phosphotriester (MPTE) linkages. The electrostatic repulsion between nDNA and its complementary strand may decrease and enhance the duplex formation. However, the methyl group of MPTEs may sterically hinder duplex formation and destabilize the duplexes. Therefore, the uses of nDNA offer additional elements of maneuvering the stability of nucleic acid duplexes. Here we used gel electrophoresis to examine the charges of the obtained nDNA, CD measurements showed that nDNA and complementary native DNA exhibited a positive peak at 275nm and a negative peak at 245nm which mainly in B-form duplexes. Surprisingly we found that nDNA destabilized the formation of DNA duplexes by using a calorimeter and Tm measurements. Slope of ∆G vs [Na+] showed that methylation of backbone phosphates leads to a lower uptake of Na+ in hybridization. We also revealed the contrary thermodynamic effects of reducing inter-stand negative-charge repulsion by shielding with sodium cations and by neutralizing the backbone negative charge with methyl groups. These later discoveries provide understanding the driving forces of formation of nucleic acid duplexes with modified nucleotides and its applications. Lastly, nDNA were applied to discriminate oligonucleotides of single-nucleotide polymorphism (SNP), Studies showed that discriminating SNP especially in GC-rich regions is often difficult because of the small difference between the melting temperature of the perfect match and the mismatch. So we chose HCV-3b which is one of the HCV genotypes as the target, by introducing different position and different density of the nDNA modification, we tried to find optimized nDNA design that can enhance the discrimination ability.
