化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

修饰和导入技术对于反义寡核苷酸的影响

合成了３′末端三个磷酸二酯键硫代修饰的针对ＤＮＡ聚合酶α的反义核苷酸ＡＳα,利用［３Ｈ］ＴｄＲ参入方法测定了ＡＳα对于ＨｅＬａ细胞ＤＮＡ复制的抑制效力。结果表明,这种修饰明显增强了寡核苷酸在含血清的细胞培养液中的稳定性。Ｌｉｐｏｆｅｃｔｉｎ能够促进ＡＳα的入胞并且增强其抑制效力。ＤＥＡＥＤｅｘｔｒａｎ也能提高ＡＳα的效力     

登革热病毒反义寡核苷酸的合成及抗病毒活性

根据碱基互补原理, 反义寡核苷酸可与特定病毒基因结合从而选择性地抑制该病毒的复制, 这是病毒性疾病治疗的新途径. 基于上述原理设计并合成了D2-04 RNA特异的6个5′末端脂肪链修饰的硫代反义寡核苷酸. 体外抗病毒活性评价显示互补于5′端起始密码、3′端重复序列和末端序列的3个反义寡核苷酸呈较强的抗病毒活性.     

cDNA微阵列与寡核苷酸芯片的制备方法

cDNA微阵列和寡核苷酸芯片是常见的合成后点样的DNA微阵列。点样方法主要是通过物理吸附或共价结合的方式将探针固定于载体上,本总结了近年来国内外献报道的cDNA微阵列制备方法;在多聚赖氨酸包被的玻璃基片表面制备cDNA微阵列;用琼脂糖包被的玻璃基片制备cDNA微阵列;在氨基或醛基修饰的玻璃基片表面制备cDNA微阵列;寡核苷酸芯片的制备方法;氨基修饰的玻片与5′末端带氨基的寡核苷酸探针通过不同的linker连接;硅烷化寡核苷酸直接点样于玻片上制成寡核苷酸微阵列;硫代寡核苷酸通过二硫键与巯基修饰的玻片连接;水凝胶芯片固定寡核苷酸。丙烯酰胺硅烷化的基片与5′丙烯酰胺修饰的寡核苷酸连接。并展望了基因芯片的应用前景。     

MALDI-TOF-MS对寡核苷酸的序列分析

通过对几种基质测定含不同碱基的寡核苷酸的灵敏度及精确度的比较,发现用混合基质α－氰基４－羟基肉桂酸（α－Ｃｙａｎｏ）／３－羟基吡啶羧酸（３ＨＰＡ）用于基质辅助激光解吸附电离飞行时间质谱中测定脱氧寡核苷酸,不仅能得到较好的分子离子峰,而且一些金属离子的加合物峰能得到有效的抑制,提高了测定的灵敏度。用３′－和５′－外切酶对脱氧寡核苷酸１２－ｍｅｒ（５′－ＡＴＧＣＡＴＡＴＧＣＡＴ－３′）进行部分降解,再进行ＭＡＬＤＩ－ＴＯＦ－ＭＳ分析,得到了完整的寡核苷酸的序列。     

抗纤维蛋白β链N端七肽抗体的制备

编码纤维蛋白β链Ｎ末端七肽（β七肽）的寡核苷酸片段,通过基因重组技术插入到金葡核酸酶（Ｐ－１蛋白）基因的５′端。在Ｐ_ＲＰ_Ｌ启动子的调控下,β七肽以融合蛋白（β七肽·Ｐ－１）的形式在Ｅ．ｃｏｌｆｉ细胞中得到高效表达。以纯化的融合蛋白为免疫原,制备出抗β七肽抗体;纤维蛋白原（ＦＧ,４ｇ／Ｌ抑制实验证明,该抗体对纤维蛋白（ＦＰ）有特异性反应。     

MALDL—TOF—MS对寡核苷酸的序列分析

通过对几种基质测定含不同碱基的寡核苷酸的灵敏度及精确度的比较,发现用混合基质α－氰基－４－羟基肉桂酸（α－Ｃｙａｎｏ）／３－羟基吡啶羧酸（３ＨＰＡ）用于基质辅助激光解吸附电离飞行时间质谱中测定脱氧寡核苷酸,不仅能得到较好的分子离子峰,而且一些金属离子的加合物峰能得到的有效的抑制,提高了测定的灵敏度,用３′和５′－外切酶对脱氧寡核苷酸１２－ｍｅｒ（５′－ＡＴＧ ＣＡＴ ＡＴＧ ＣＡＴ－３′）进行部分     

鱼类LZF-IDNA指纹探针的制备

利用Ｏｌｉｇｏ１０００ＤＮＡ合成仪（Ｂｅｃｋｍａｎ）合成了长度为４５ｂｐ的寡核苷酸单链,经纯化后用同位素γ－３２Ｐ－ＡＴＰ作５′末端标记后,制备成鱼类ＬＺＦ－ＩＤＮＡ指纹探针。通过对鱼类的群体实验、亲子鉴定实验、组织细胞的稳定性实验和鱼类种类的适用范围实验后,测得：（１）ＬＺＦ－ＩＤＮＡ指纹探针属多位点寡核苷酸探针;（２）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类种群中的鉴别机率为９．２３×１０－１６;（３）ＬＺＦ－ＩＤＮＡ指纹探针在鱼类亲子鉴定实验中的父系概率为０．９９９９６２;（４）ＬＺＦ－ＩＤＮＡ指纹探针,是一种稳定的,既具有个体识别能力,又具有一定种属特异性的、适用于鱼类ＤＮＡ指纹图研究的基因指纹探针。     

反义寡核苷酸药物癌泰得的定性分析方法研究

 癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .     

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), and CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA.TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A.T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in two different structures are reported.     

Facile preparation of nuclease resistant 3' modified oligodeoxynucleotides.

An efficient chemical procedure for the immobilization of carboxylate containing conjugate groups onto controlled pore glass (CPG) is described. The derivatized supports were used in the automated synthesis of an oligodeoxynucleotide (20-mer ODN) containing a 3' phosphodiester linked hexanol, aminohexyl, acridine, or cholesterol group. The stability of the oligomer in a hepatoma cell culture was found to be prolonged two to three fold by the presence of any of the 3' tails. By contrast, an aminohexyl group appended to the 5' terminus of the ODN only marginally improved its nuclease resistance. These data support the notion that antisense ODNs are primarily degraded by 3' exonucleases. Introduction of simple 3' tails which incorporate a normal phosphodiester linkage can increase ODN stability by interfering with these enzymes.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Synthesis, gene-silencing activity and nuclease resistance of 3'-3'-linked double short hairpin RNA

To improve the nuclease resistance of siRNA while reducing its induction of an innate immune response and maintaining its biological activity for possible therapeutic application, we designed and synthesized a series of double short hairpin RNAs (dshRNAs). Each dshRNA consisted of two identical short hairpin RNAs (shRNAs) linked at their 3' ends by glycerol. The dshRNAs were synthesized on a glycerol-derivatized solid support from amidites with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxyl protecting group. Synthesis was carried out in a single run on a DNA/RNA synthesizer, without the need for enzymatic ligation. The dshRNAs showed structure-dependent gene-silencing activity at the protein level, and dshRNAs in which the 3' end of the two sense regions were linked showed especially high activity. Inclusion of 2'-O-methyluridine residues in the loop region was associated with 1.6- to 2.4-fold lower induction of interferon-α than was siRNA, without loss of gene-silencing activity. dshRNA also showed higher exonuclease resistance than siRNA or canonical shRNA. Our studies provide a new approach to gene silencing based on the concept of linking the 3' end of the sense regions of two shRNA molecules to form a double shRNA.     

Stability of single-nucleotide bulge loops embedded in a GAAA RNA hairpin stem

Forty-six RNA hairpins containing combinations of 3' or 5' bulge loops and a 3' or 5' fluorescein label were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each hairpin were determined. The bulge loops were of the group I variety, in which the identity of the bulge is known, and the group II variety, in which the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The fluorescein label at either the 3' end or 5' end of the hairpin did not significantly influence the stability of the hairpin. As observed with bulge loops inserted into a duplex motif, the insertion of a bulge loop into the stem of a hairpin loop was destabilizing. The model developed to predict the influence of bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability. Specifically, the influence of the bulge is related to the stability of the hairpin stem distal from the hairpin loop.     

A new Vibrio fischeri lux gene precedes a bidirectional termination site for the lux operon.

The DNA downstream of the lux structural genes in the Vibrio fischeri lux operon has been sequenced and a new lux gene (luxG) has been identified. A hairpin loop that begins with a poly(A) region and ends with a poly(T) region and thus can function as a bidirectional termination site for luxG and a convergent gene is located immediately downstream of luxG. 3' S1 nuclease mapping has demonstrated that the luxG mRNA was induced in a cell-density-dependent fashion consistent with it being part of the lux system and that the lux mRNA terminated immediately after the hairpin loop. The mRNA coded by an open reading frame convergent to luxG on the complementary strand was also shown by S1 nuclease mapping to overlap the lux mRNA for at least 20 nucleotides before termination. Expression of DNA containing the hairpin loop, placed between a strong promoter and a reporter gene and transferred by conjugation into luminescent bacteria, demonstrated the very high efficiency of termination by this hairpin loop oriented in either direction. These results also demonstrate that the organization of the genes at the 3' ends of the lux operons of V. fischeri and V. harveyi has clearly diverged.     

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.Abbreviations The hairpin oligonucleotides are indicated by hp hairpin followed by the loop sequence, starting at the 5'-end, in parenthesis; d for deoxy is omitted for clarity     

Multinuclear NMR studies of DNA hairpins. 1. Structure and dynamics of d(CGCGTTGTTCGCG)

The solution structure of the hairpin formed by d(CGCGTTGTTCGCG) has been examined in detail by a wide variety of NMR techniques. The hairpin was characterized by proton NMR to obtain interproton distances and torsion angle information. An energy-minimized model was constructed that is consistent with these data. The hairpin consists of a B-DNA stem of four C-G base pairs and a loop region consisting of five unpaired bases. Three bases in the 5' of the loop are stacked over the 3' end of the stem, and the other two bases in the 3' of the loop are stacked over the 5' end of the stem. The phosphorus NMR spectrum revealed a phosphate in the stem region with an unusual conformation, and two phosphates, P9 and P10, were found to undergo intermediate exchange between conformations. The hairpin was also synthesized with a carbon-13 label in each of the thymidine C6 carbons, and relaxation measurements were performed to determine the extent of internal motions in the loop region. The loop bases are more flexible than the stem bases and exhibit subnanosecond motions with an amplitude corresponding to diffusion in a cone of approximately 30 degrees.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Novel DNA/polymer conjugate for intelligent antisense reagent with improved nuclease resistance

Antisense technology provides an effective strategy to inhibit synthesis of the gene product. We prepared a novel antisense reagent comprised of oligodeoxynucleotides (ODN) and a thermo responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). The conjugate inhibited gene expression in a dose-dependent manner. The ODN-PNIPAAm conjugate demonstrated excellent resistance to S1 nuclease. In particular, PNIPAAm-modified antisense ODN at the 3',5'-ends of the ODN provided complete resistance against nuclease at 37 degrees C, which is above the phase transition temperature of the PNIPAAm side chain. These characteristics of the conjugate suggest it may have potential for use in a new gene delivery system as part of an antisense strategy.