| 摘要: | 反義寡核苷酸（Antisense oligonucleotides, ASOs）是設計用來特異性地與信使RNA（messenger RNA, mRNA）互補序列結合的合成單鏈DNA或RNA分子。然而，未經修飾的DNA ASOs通常顯示出低親和力和生物不穩定性。在本研究中，我們引入了一種稱為中性DNA（neutralized DNA, nDNA）的DNA ASO。這些ASOs是通過對DNA結構內的磷酸二酯鍵的磷酸基進行甲基化修飾，化學合成的位點特異性磷酸甲基三酯（methyl phosphotriester, MTPE）鍵。DNA磷酸骨架的負電荷被中和，從而減少了核酸雙鏈之間的靜電排斥，並提高了親和力和穩定性。為了將nDNA ASOs輸送到細胞中，我們使用了多孔性二氧化矽奈米粒子（mesoporous silica nanoparticles, MSN）作為載體。
在癌症細胞中，部分致癌基因（Oncogene）因為細胞基因突變導致過量表達，例如c-Myc、Survivin，造成細胞代謝方式改變以及異常增殖。此外，Greider和Blackburn在1985年發現的端粒酶（telomerase），通過維持染色體端粒（telomere）的長度，使細胞變得永生。端粒酶在許多癌細胞中表達，但在正常人體體細胞中幾乎不表達。因此，這些在癌細胞特有的過量表達mRNA或蛋白質，成為癌症治療中的良好標靶。
本研究分為兩部分。在第一部分中，為了評估nDNA ASO對長鏈RNA的調控能力，我們使用表達報告基因（reporter gene）增強綠色螢光蛋白（enhanced green fluorescent protein, eGFP）mRNA的直腸癌細胞HCT-116 eGFP，評估不同數量的MTPE修飾的nDNA抑制eGFP mRNA的效率。隨後，我們選擇人類端粒酶逆轉錄酶mRNA（human telomerase reversed transcriptase mRNA, hTERT mRNA）與人類端粒酶核酸模板（Human Telomerase RNA Template, hTR）作為內源性基因抑制的目標。然而，長鏈RNA分子中存在二級、三級結構，這些立體結構可能影響nDNA ASO對目標RNA抑制能力。在本研究中，eGFP nDNA ASO序列參考過去研究所使用的siRNA序列，hTERT nDNA ASO參考鎖核酸（Locked nucleic acid, LNA） gapmer序列，而hTR nDNA ASO則是參考磷硫酸脂（Phosphorothioates, PS）ASO序列，篩選出合適的nDNA ASO序列。
nDNA ASO藉由序列互補與目標RNA結合，使目標mRNA無法被核醣體轉譯出蛋白質。結果顯示，當目標序列是eGFP mRNA和hTERT mRNA，nDNA ASO皆無法有效地抑制；而目標序列是hTR時，nDNA ASO可以成功抑制hTR逆轉錄。這些siRNA和核酸類似物的RNA干擾（RNA interference, RNAi）機制不同，其分子對目標核酸序列親和力不同，導致對於相同核酸序列之抑制能力也隨之不同。
在本研究第二部分中，我們藉由調整hTR nDNA ASO的nDNA修飾位置與hTR nDNA ASO序列長度，來看對於hTR逆轉錄抑制能力之影響。在nDNA修飾位置上，我們發現當nDNA修飾在對應hTR環結構上，展現出更佳的抑制能力，此結果也呼應Buck的nDNA抑制HIV-1 RNA研究。進一步我們根據hTR環結構相對位置調整nDNA ASO長度，發現延長nDNA ASO並包覆環結構反而降低nDNA ASO抑制能力；而將nDNA ASO序列縮短並只包覆環結構，則能夠提升nDNA ASO的抑制能力，並且隨著nDNA修飾數量越多，抑制能力越好。
本研究結果揭露利用部分磷酸根甲基化nDNA ASO在HCT-116細胞與HCT-116 eGFP細胞內調控長鏈RNA，其目標RNA結構如何影響nDNA ASO調控能力，並初步比較相同目標序列siRNA、LNA gapmer ASO與PS ASO，nDNA ASO對這些目標序列的抑制能力，期望能用作未來用於其他致癌基因治療之nDNA ASO設計上參考。
;ASOs are synthetic single-stranded DNA or RNA molecules designed to bind specifically to complementary sequences of messenger RNA (mRNA). However, unmodified DNA-based ASOs often exhibit low affinity and bio-instability. In this study, we introduced a class of DNA-based ASO called neutralized DNA (nDNA). These ASOs are chemically synthesized with site-specific internucleoside methyl phosphotriester (MPTE) linkages, achieved by methylating the phosphate groups of the phosphodiester bonds within the DNA structure. The negatively charged DNA phosphodiester backbone is neutralized, thus reducing electrostatic repulsion between nucleic acid double strands and leading to enhanced affinity and stability. To deliver nDNA ASOs into cells, mesoporous silica nanoparticle (MSN) was used as the carrier.
In cancer cells, certain oncogenes are overexpressed due to genetic mutations, such as c-Myc and Survivin, leading to changes in cell metabolism and abnormal proliferation. Additionally, telomerase, discovered by Greider and Blackburn in 1985, maintains chromosome telomere length, allowing cells to become immortal. Telomerase is expressed in many cancer cells but hardly in normal human somatic cells. Therefore, these overexpressed mRNAs specific to cancer cells become good targets for cancer treatment.
This study is divided into two parts. In the first part, to evaluate the regulatory ability of nDNA ASO on long-chain mRNA, we used HCT-116 eGFP, HCT-116 cells expressing reporter gene enhanced green fluorescent protein (eGFP) mRNA, to assess the efficiency of different numbers of MTPE-modified nDNA in inhibiting eGFP mRNA. Subsequently, we selected human telomerase reverse transcriptase mRNA (hTERT mRNA) and human telomerase RNA template (hTR) as targets for endogenous gene inhibition. However, the secondary and tertiary structures in long-chain RNA molecules might affect the inhibitory ability of nDNA ASO on target RNA. In this study, the eGFP nDNA ASO sequence was referenced from siRNA sequences used in previous research, the hTERT nDNA ASO was based on Locked Nucleic Acid (LNA) gapmer sequences, and the hTR nDNA ASO was referenced from phosphorothioate (PS) ASO sequences, selecting suitable nDNA ASO sequences. The different mechanisms of RNAi-induced enzyme degradation and varying molecular affinities for the target sequences lead to different inhibitory capabilities of RNAi on the same sequence.
Previous research by Buck on HIV-1 RNA inhibition indicated that nDNA sequences have better inhibitory effects on RNA sequences with loop secondary structures, and the more free bases on the loop, the higher the nDNA inhibition capacity. In the second part, we used RNA fold analysis to examine the hTR structure and found that modifying nDNA at positions corresponding to the hTR loop structure can enhance inhibition. However, extending the ASO 5′ end to fully cover the hTR loop structure reduced the inhibitory effect of nDNA ASO on hTR. We hypothesize that extending the ASO sequence leads to more DNA-RNA hybrid formation, resulting in more B-form to A-form transitions and decreased sequence affinity. Shortening the ASO 3′ end to cover only the hTR loop target sequence increases the nDNA ASO inhibitory ability, and more nDNA modifications further improve the nDNA ASO′s ability to inhibit hTR.
The results provide insights into the design considerations of nDNA ASOs, suggesting that the selection of target sequences should avoid regions rich in secondary and tertiary structures or RNA-binding protein regions. By using RNA fold analysis and referencing previously successful ASO sequences, suitable nDNA ASO sequences can be selected. Once the appropriate nDNA ASO sequences are chosen, the sequence length and nDNA modification locations relative to the target sequence loop structure can influence the nDNA ASO′s inhibitory ability. This information can reduce the labor and time costs in future nDNA ASO design efforts in our laboratory, leading to the discovery of effective nDNA ASOs for inhibiting various oncogenes. |
