RNA干扰技术的原理与应用

RNA干扰(RNA interference,RNAi)是由双链RNA(double-stranded RNA,dsRNA)所引起的序列特异性基因沉默,是真核生物中一种非常保守的机制,它与协同抑制(cosuppression)、转座子沉默(transposon silencing)以及发育等许多重要的生物学过程密切相关。RNA干扰依赖于小干扰RNA(Small interference RNA,siRNA)与靶序列之间严格的碱基配对,具有很强的特异性,涉及众多基因和蛋白复合物,构成了一个以小RNA为核心的真核基因表达调控系统,它可以在染色质水平、转录水平、转录后水平和翻译水平参与基因表达的调节。RNA干扰技术为人们迅速、准确的剖析基因的功能,分析基因之间错综复杂的联系和相互作用提供了极为有用的工具,同时也为人们预防和治疗癌症和病毒疾病提供了新的思路。     

非编码RNA的生物信息学研究方法:RNA结构预测及其应用

近10多年来的研究逐步揭示了RNA的各种生物学功能。RNA不仅是信息从DNA传递到蛋白质的中间体,还直接参与基因沉默、表观遗传学修饰等生物学过程。单链的RNA在体内通过碱基配对折叠成一定的二级结构。介绍了现在预测RNA二级结构的主要算法及其应用,其中包括基于热力学、同源比对和统计学习的各种算法,以及如何引入实验数据辅助预测。二级结构预测算法被广泛用于寻找RNA功能单元和预测新非编码RNA等各种问题。如何利用高通量实验数据帮助结构预测,探索长非编码RNA功能,研究RNA与蛋白质相互作用,是RNA二级结构预测算法和应用的一些前沿方向。     

RNA三维结构的测定:基于冷冻电子显微镜技术的新视角

RNA在人类生物学和疾病功能障碍中发挥重要作用.对于绝大多数RNA分子而言,复杂的高级结构是其发挥功能的先决条件.了解RNA的三维结构对于研究非编码RNA功能、致病机理以及开发靶向药物至关重要.然而,由于RNA的固有灵活性,实验测定其三维结构的难度较大.冷冻电子显微镜技术(冷冻电镜, cryo-EM)得益于探测器技术和软件算法的快速发展,且不受核磁共振对小分子量及X射线晶体学对结晶的要求所制约,近年来在其他结构分析方法中脱颖而出.本文概述近年来以冷冻电子显微镜技术为核心联用其他技术(如核磁共振、晶体学和理论建模等)解析RNA三维结构的研究进展,以促进RNA结构研究和RNA靶向药物设计的发展.     

反义RNA技术

反义RNA(antisence RNA)是一种与特异mRNA互补的RNA分子,它通过配对碱基间氢键作用与对应的RNA形成双链复合物,抑制RNA的翻译过程。利用人工合成或生物体合成特定互补RNA片段(或其化学修辞产物)抑制或封闭基因表达技术,称为反义RNA技术。本文综述了反义RNA作用机理,合成途径,并展示了该技术在研究基因功能,防治肿瘤、遗传病以及人工免疫等方面广阔的应用前景。一、反义RNA抑制基因表达机理许多实验证明,原核类生物如细菌、粘菌,     

反义技术研究进展

反义技术利用DNA或RNA分子通过Watson Crick碱基配对原则与目的基因的mRNA互补结合 ,通过各种机制使其降解或抑制其编码蛋白的翻译 ,从而抑制目的基因的表达。与基因敲除(geneknockout)等功能缺失性研究方法相比 ,反义技术具有投入少 ,周期短 ,操作简单等优点 ,因此受到了广泛的关注。对几种常用反义技术的研究进展及存在的问题进行概述。     

G-四链体:核酸的“新”结构

<正>核酸(包括DNA和RNA)是体内最重要一类生物大分子,DNA主要作为遗传信息载体,而RNA则作为信息中介分子、调节因子和酶等发挥多重生物学功能。核酸分子空间结构形式复杂、可变,可采用多种构象,在体内DNA主要为双螺旋结构,而RNA采取单链为主局部双螺旋结构,此外还存在一些特殊结构,如Z-DNA和G-四链体(G-quadruplex)等,这些结构对保证基因组完整性、基因表达和调控都具有重要作用。由于结构     

细胞核内RNA干扰途径研究进展

RNA干扰(RNAi)是双链RNA分子在mRNA水平关闭相应序列基因表达的过程。RNAi的分子机制及功能仍然有待深入研究与阐明,但已经在基因组结构与功能研究中得到广泛运用。我们简要综述RNAi的作用机制,及已发现的细胞核内干扰途径——RNA指导的DNA甲基化、异染色质、DNA切除和减数分裂性沉默等相关研究进展。     

非编码RNA与基因表达调控

近年来,随着对基因组的深入研究,发现真核生物中存在许多形态和功能各异的非编码RNA分子,这类RNA分子并不表达蛋白质,但它们在基因转录水平、转录后水平及翻译水平起了重要的调控作用。具有调控作用的RNA分子种类非常丰富,如长链非编码RNA(long non-coding RNA,lncRNA)、miRNA、PIWI相互作用RNA(PIWI-interacting RNA,piRNA)、内源性小干扰RNA(endogenous small interfering RNA,endo-siRNA)、竞争性内源RNA(competitive endogenous RNA,ceRNA)等,它们使基因表达过程更为丰富、严谨和有序。本文综述几类典型的非编码RNA对基因表达的调节作用,以助于理解细胞中RNA分子调节网络的功能和机制。     

DNA折纸纳米载体在药物递送中的应用

DNA纳米技术是基于沃森克里克碱基配对原则产生可编程核酸结构的技术。因其具有高精度的工程设计、前所未有的可编程性和内在的生物相容性等特点，运用该技术合成的纳米结构不仅可以与小分子、核酸、蛋白质、病毒和癌细胞相互作用，还可以作为纳米载体，递送不同的治疗药物。DNA折纸作为一种有效的、多功能的方法来构建二维和三维可编程的纳米结构，是DNA纳米技术发展的一个里程碑。由于其高度可控的几何形状、空间寻址性、易于化学修饰，DNA折纸在许多领域具有巨大的应用潜力。本文通过介绍DNA折纸的起源、基本原理和目前进展，归纳总结了运用DNA折纸进行药物装载和释放的方式，并基于此技术，展望了今后的发展趋势以及所面临的机遇和挑战。     

与生物体发育相关的非编码RNA及其作用机理

非编码RNA是一类没有开放阅读框、不能翻译成为蛋自质的RNA分子。在哺乳动物中,它们主要是指微小RNA、小干扰RNA、PIWI互作RNA和其他一些反义转录本等。它们在生物体内广泛存在,通过RNA干扰、基因沉默、基因印迹和DNA甲基化等机制调控着基因的表达。非编码RNA增加了真核细胞调控网络的复杂性,也为科学地解释一些现象提供了新的途径。     

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.     

Positional effect of single bulge nucleotide on PNA(peptide nucleic acid)/DNA hybrid stability.

We report positional effect of bulge nucleotide on PNA/DNA hybrid stability. CD spectra showed that PNA/DNA hybrids required at least seven base pairings at a stem region to form a bulged structure. On the other hand, DNA/DNA could form bulged structure when there are only four base pairings adjacent to the bulge nucleotide. We discuss why PNA requests such a many base pairings to form bulged structure from a nearest neighbor standpoint.     

Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.     

Rearrangement of a stable RNA secondary structure during VS ribozyme catalysis

The Neurospora VS ribozyme recognizes and cleaves a substrate RNA that contains a GC-rich stem loop. In contrast to most RNA secondary structures that are stable during tertiary or quaternary folding, this substrate undergoes extensive ribozyme-induced rearrangement in the presence of magnesium in which the base pairings of at least seven of the ten nucleotides in the stem are changed. This conformational switch is essential for catalytic activity with the wild-type substrate and creates a metal-binding secondary structure motif near the cleavage site. Base pair rearrangement is accompanied by bulging a cytosine from the middle of the stem, indicating that ribozymes may perform base flipping, an activity previously observed only with protein enzymes that modify DNA.     

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

The 3′ end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3′ translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3′ end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3′ end, which causes structural changes that potentiate the shift between translation and replication.     

Energy hyperspace for stacking interaction in AU/AU dinucleotide step: Dispersion‐corrected density functional theory study

Double helical structures of DNA and RNA are mostly determined by base pair stacking interactions, which give them the base sequence‐directed features, such as small roll values for the purine–pyrimidine steps. Earlier attempts to characterize stacking interactions were mostly restricted to calculations on fiber diffraction geometries or optimized structure using ab initio calculations lacking variation in geometry to comment on rather unusual large roll values observed in AU/AU base pair step in crystal structures of RNA double helices. We have generated stacking energy hyperspace by modeling geometries with variations along the important degrees of freedom, roll, and slide, which were chosen via statistical analysis as maximally sequence dependent. Corresponding energy contours were constructed by several quantum chemical methods including dispersion corrections. This analysis established the most suitable methods for stacked base pair systems despite the limitation imparted by number of atom in a base pair step to employ very high level of theory. All the methods predict negative roll value and near‐zero slide to be most favorable for the purine–pyrimidine steps, in agreement with Calladine's steric clash based rule. Successive base pairs in RNA are always linked by sugar–phosphate backbone with C3′‐endo sugars and this demands C1′–C1′ distance of about 5.4 Å along the chains. Consideration of an energy penalty term for deviation of C1′–C1′ distance from the mean value, to the recent DFT‐D functionals, specifically ωB97X‐D appears to predict reliable energy contour for AU/AU step. Such distance‐based penalty improves energy contours for the other purine–pyrimidine sequences also. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 107–120, 2014.     

Diverse self-association properties within a family of phage packaging RNAs

The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage''s DNA genome. The pRNA forms higher-order multimers by intermolecular “kissing” interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA''s junction, we propose a model for how different junctions affect self-association.