siRNA和miRNA介导的基因沉默（导读版） 下载 pdf 百度网盘 epub 免费 2025 电子书 mobi 在线

RNA干扰已经成为抑制基因表达和治疗药物发展的重要方法，然而该方法在输送、稳定性以及抑制不相干基因表达的脱靶效应和激活先天免疫等方面仍然存在着很大问题。siRNA和miRNA介导的基因沉默：从实验室到临床应用（导读版）中，研究人员探讨了siRNA的设计、表达、输送、体内成像、降低副作用及促进其在病人体内的应用等*进展。作为极其成功的《分子生物学方法》系列丛书的分卷，各章节中的相关主题着重提供易用且*的信息，包括在体外和体内极具应用前景的新输送载体的构想和策略等详细实验步骤，治疗相关靶基因的验证及参与miRNA功能、生物发生和干扰病毒感染的相关分子。siRNA和miRNA介导的基因沉默：从实验室到临床应用（导读版）为那些对RNA干扰、基因调节和新治疗方法感兴趣的人员提供帮助。

前言

撰稿人

第1章 运用统计学和聚类分析的方法选择有效的siRNA序列

第2章 破解先天免疫识别siRNA的密码

第3章 哺乳动物细胞中反义寡核苷酸和siRNA的靶向输送

第4章 用于局部及全身性治疗的寡核苷酸siRNA的导入

第5章 siRNA转导及沉默成像

第6章 磁转染及其在体外导入siRNA潜能的研究进展

第7章 TransKingdom RNAi(tkRNAi)在体外和体内的基因沉默

第8章 用细菌输送siRNA:一个实体瘤治疗的新方法

第9章 锁核苷酸修饰的siRNA的治疗潜能:通过对siRNA序列的化学修饰来减少脱靶效应

第10章 miRNA和shRNA的pSM155和pSM30表达载体

第11章 用siRNA靶向癌基因

第12章 用siRNA靶向间质与癌细胞之间的相互作用

第13章 siRNA介导的靶向基质金属蛋白酶、尿激酶纤维蛋白溶酶原激活剂及其受体的治疗潜质

第14章 用RNA干扰方法在体外和体内沉默人神经胶质瘤细胞中的HIF-1a基因

第15章 运用RNA干扰的方法验证参与端粒维持和抗凋亡相关的癌基因是否可作为癌症治疗的靶标

第16章 应用经化学修饰的第二代鼻内siRNA治疗呼吸道病毒疾病

第17章 抗1型人类免疫缺陷病毒的siRNA应用进展

第18章 microRNA通路的蛋白组分与人类疾病

第19章 内含子介导的RNA干扰和microRNA生物发生

第20章 HIV-1与microRNA通路的复杂关系

第21章 应用合成的microRNA靶向神经胶质瘤相关抗原蛋白-1

第22章 运用siRNA直接抑制BCR-A BL转录产物的方法来靶向治疗耐伊马替尼的慢性髓细胞白血病患者

索引

〔挪〕西乌德（Sioud，M.）

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Chapter 1

Methods for Selecting Effective siRNA Sequences by Using Statistical and Clustering Techniques

Shigeru Takasaki

Abstract

Short interfering RNAs (siRNAs) have been widely used for studying gene functions in mammalian cells but vary markedly in their gene-silencing efficacy. Although many design rules/guidelines for effective siRNAs based on various criteria have been reported recently, there are only a few consistencies among them. This makes it difficult to select effective siRNA sequences targeting mammalian genes. This chap-ter first reviews the reported siRNA design guidelines and clarifies the problems concerning the current guidelines. It then describes the recently reported new scoring methods for selecting effective siRNA sequences by using statistics and clustering techniques such as the self-organizing map (SOM) and the radial basis function (RBF) network. In the proposed three methods, individual scores are defined as a gene degradation measure based on position-specific statistical significances. The effectiveness of the methods was confirmed by evaluating effective and ineffective siRNAs for recently reported genes and comparison with other reported scoring methods. The sizes (values) of these scores are closely correlated with the degree of gene degradation, and the scores can easily be used for selecting high-potential siRNA candidates. The evaluation results indicate that the proposed new methods are useful for selecting siRNA sequences targeting mammalian mRNA sequences.

Key words: siRNA design , RNA interference , gene silencing , SOM classification , statistical significance , RBF network .

1. Introduction

Although RNA interference (RNAi) has been successfully used for studying gene functions in both plants and invertebrates, many practical obstacles need to be overcome before it becomes an established tool for use in mammalian systems (1? 6) . One of the important problems is designing effective short interfering RNA (siRNA) sequences for target genes. The siRNA responsible for

M. Sioud (ed.), Methods in Molecular Biology, siRNA and miRNA Gene Silencing, vol. 487 . Humana Press, a part of Springer Science + Business Media, LLC 2009 DOI: 10.1007/978-1-60327-547-7_1

1

Takasaki

RNA interference varies markedly in its gene-silencing efficacy in mammalian genes, where the gene-silencing effectiveness depends very much on the target sequence positions (sites) selected from the target gene (7,8). Since different siRNAs synthesized for vari-ous positions induce different levels of gene silencing, the selec-tion of the target sequence is critical for the effectiveness of the siRNA. We therefore need useful criteria for gene-silencing effi-cacy when we design siRNA sequences (9,10).

Zamore et al. and Jayasena et al. showed that the 5 ′ end of the antisense strand might be incorporated into the RNA-induced silencing complex (RISC). Strand incorporation may depend on weaker base pairing and thus an A?T terminus may lead to more strand incorporation than a G?C terminus (11,12). Other factors reported to be related to gene-silencing efficacy are GC content, point-specific nucleotides, specific motif sequences, and secondary structures of mRNA. Several siRNA design rules/guidelines using efficacy-related factors have been reported (13 ?17).

Although the positional nucleotide characteristics for siRNA designs seem to be the most important factor determining effec-tive siRNA sequences, there are few consistencies among the reported rules/guidelines (18?23). This implies that these rules/ guidelines might result in the generation of many candidates and thus make it difficult to extract a few for synthesizing siR-NAs. Furthermore, there is in RNAi a risk of off-target regula-tion: a possibility that the siRNA will silence other genes whose sequences are similar to that of the target gene. When we use gene silencing for studying gene functions, we have to first some-how select high-potential siRNA candidate sequences and then eliminate possible off-target ones (24).

This chapter first reviews the reported siRNA design guide-lines and clarifies the problems concerning the reported guide-lines. It then describes the recently reported new scoring methods for selecting effective siRNA sequences by using statistical and clustering techniques (25?32).

In the statistical method, many effective siRNA sequences are examined in the literature (31), because it can be hypoth-esized that position-specific nucleotides play important roles in gene-silencing efficacy. If specific features of nucleotide frequen-cies appeared in many effective siRNAs, they mean the positional nucleotide characteristics for siRNA designs. The features of nucleotide frequencies at individual positions are then analyzed by using the statistical significance test. As these features can be considered as new guidelines, a measure (score) for select-ing effective siRNA candidates is defined based on the positional features of specific significant nucleotides. The effectiveness of the proposed measure was confirmed by comparing the com-puted scores with those of the recently reported other selection methods (28,29,31).

2. RNA Interfer-ence and siRNA Sequence Selec-tion Problem

2.1. RNA Interference

Methods for Selecting Effective siRNA Sequences

The chapter then describes how to extract individual nucle-otide features from many effective siRNA sequences by using mathematical clustering techniques ? the SOM and the RBF network (see later Sects. 3.2.1 and 3.2.2) (25?27). In the SOM-based clustering method, siRNA classification from many effective siRNAs is first described. It is then shown how posi-tional nucleotide features are extracted from the classified groups and is demonstrated how the extracted features are integrated as a measure (score). It is finally confirmed that the SOM method is effective by evaluating the relations between the scores and effec-tive/ineffective siRNAs reported in the literature and comparing them with those of other reported scoring methods (30,33).

In the RBF-network-based method, after the siRNA classi-fication is carried out by using the RBF network (25,26), the preferred and unpreferred nucleotides for effective siRNAs at individual positions are determined by significance testing and are used to calculate a score that measures a sequence’s poten-tial for gene degradation. The effectiveness of the proposed scor-ing method was then confirmed by using it to evaluate RNA sequences recently reported to effectively or ineffectively sup-press the expression of various genes (see later subsection) and comparing it with other scoring methods (32,33).

As a result of various evaluations, it is found there are good correlations between the sizes (values) of the proposed individual scores and the effectiveness and ineffectiveness of the recently reported siRNA sequences. The evaluation results indicate that the three methods would be useful for selecting siRNA sequences for mammalian genes.

RNA interference (RNAi) is a phenomenon that silences gene expression by introducing double-stranded RNA (dsRNA) homologous to the target mRNA (1). After this phenomenon was discovered in the nematode Caenorhabditis elegans, it gradually became clear that similar phenomena occur in the cells of plants, fungi, and mammals (1?6). RNAi has been reported to result from the following sequence of events (2,5,6). Long dsRNA is first cleaved into siRNA species by an RNAase III enzyme, Dicer. These siRNAs are then incorporated into an RNA-induced silenc-ing complex (RISC), where the duplex siRNA is unwound so the antisense strand can guide RISC to the target mRNA having the complementary sequence. Finally, the target mRNA is cleaved at a single site in the center of the duplex region between the guide siRNA and the target mRNA (28). Among the events that are

Takasaki

2.2. siRNA Sequence Selection Problem

2.2.1. Related Works Regarding the siRNA Sequence Selection Problem

2.2.2. The Reported Guide-lines for siRNA Sequence Design

still unclear, however, are the mechanism of the target mRNA cleavage and the mechanism by which the center of the duplex region in the RISC is identified. Furthermore, although RNAi has been widely used for studying gene functions, the effectiveness with which the genes in mammalian cells can be silenced this way depends very much on target sequence positions (sites) selected from the target gene. That is, different siRNAs synthesized from various positions induce different levels of gene silencing. This indicates that the selection of the target sequence position (site) is critical for the effectiveness of the siRNA (3?6).

To use RNAi as a biological tool for mammalian cell experiments, we first need to identify target sequences causing gene degrada-tion. They have so far been identified by using a trail-and-error method (3,8), but siRNAs extracted from different regions of the same gene have varied remarkably in their effectiveness. The difficulty of using the trail-and-error method to select tar-get sequences causing gene silencing increases when the coding regions are long, as they are in mammalian cells. This is because the larger the number of candidates becomes, the more difficult it is to get gene-silencing candidates.

The earliest guidelines for siRNA sequence design were proposed by Elbashir et al. (4,8,40). They suggested that synthesizing siRNA duplexes of 21 nucleotides (nt) length ? 19 nt base-paired sequence with 2 nt 3′ overhang at the ends ? mediates efficient cleavage of the target mRNA. Their rules are summarized as follows.

(1)

Select the target region from the open reading frame (ORF) of a given cDNA sequence preferably 50?100 nt down-stream of the start codon. Avoid 5 ′ or 3′ untranslated regions (UTRs) or regions close to the start codon as these may be richer in regulatory protein-binding sites.

(2)

Search for sequences 5 ′-AA(N19)UU, where N is any nucleotide, in the mRNA sequence and choose those with approximately 50% GC content. Highly G-rich sequences should be avoided because they tend to form G-quar-tet structures. If there are no 5 ′-AA(N19)UU motifs present in the target mRNA, search for 5 ′-AA(N21) or 5′-NA(N21), and synthesize the sense siRNA as 5′-(N19) TT and the antisense siRNA as 5′-(N′19)TT, where N ′19 denotes the reverse complement sequence of N19 and T indicates 2′-deoxythymidine.

(3)

Blast-search the selected siRNA sequences against EST libraries or mRNA sequences of the respective organism to ensure that only a single gene is targeted.

(4)

It may be advisable to synthesize several siRNA duplexes to control for the specificity of the knockdown experiments; those siRNAs duplexes that are effective for silencing should produce exactly the same phenotype. Furthermore, a non-specific siRNA duplex may be needed as control.

(5)

If the siRNA does not work, first verify that the target sequence and the cell line used are derived from the same organism. Finally, make sure that the mRNA sequence used for selection of siRNA duplexes is reliable; it could contain sequencing errors, mutations, or polymorphisms.

Methods for Selecting Effective siRNA Sequences

After that, many siRNA design guidelines/rules were reported as follows. Reynolds et al. analyzed 180 siRNAs systematically, targeting every other position of two 197-base regions of fire-fly luciferase and human cyclophilin B mRNA (90 siRNAs per gene), and reported the following eight criteria for improving siRNA selection (18).

G1 (Reynolds et al.) eight criteria: (1) G/C content 30?52%,

(2) at least 3 As or Ts at positions 15?19, (3) absence of internal repeats, (4) an A at position 19, (5) an A at position 3, (6) a T at position 10, (7) a base other than G or C at position 19, and (8) a base other than G at position 13.

Ui-Tei et al. examined 72 siRNAs targeting six genes and reported four rules for effective siRNA designs (19). They are summarized as follows.

G2 (Ui-Tei et al.) four rules: (1) A or T effective and G or C ineffective at position 19, (2) G or C effective and A or T ineffec-tive at position 1, (3) at least 5 T or A residues from positions 13 to 19, and (4) no GC stretch more than 9 nt long.

Amarzguioui and Prydz analyzed 46 siRNAs targeting four genes and reported the following six rules for effective siRNA designs based on their literature (20).

G3 (Amarzguioui and Prydz) six rules: (1) G or C positive and T negative at position 1, (2) A positive at position 6, (3) T negative at position 10, (4) T positive at position 13, (5) C positive at position 16, and (6) A or T positive and G negative at position 19.

Jagla et al. tested 601 siRNAs targeting one exogenous and three endogenous genes and reported four rules as follows (22).

G4 (Jagla et al.): (1) A or T positive at position 19, (2) A or T positive at position 10, (3) G or C positive at position 1, and (4) more than three A/Ts between positions 13 and 19.

Hsieh et al. examined 138 siRNAs targeting 22 genes and reported the following position-specific characteristics (21).

G5 (Hsieh et al.): (1) T positive and G negative at position 19, (2) C or G positive and A or T negative at position 11, (3) G positive at position 16, (4) A positive at position 13, and (5) C negative at position 6.

Takasaki

The above previous works for positional characteristics in siRNA designs are summarized in Table 1.1 a.

Other scoring, screening, and designing methods for func-tional siRNAs have also been reported recently. Chalk et al. reported seven rules (“Stockholm rules”) based on thermody-namic properties. They are (1) total hairpin energy

Huesken et al. reported the screen method of functional siR-NAs by using an artificial neural network (23). This network was first trained by 2182 randomly selected siRNAs targeted to 34 genes and was used in the design of a genome-wide siRNA col-lection with two potent siRNAs per gene.

Teramoto et al. and Ladunga reported functional siRNA selection methods using support vector machine (SVM) (14,34). Teramoto et al. used generalized string kernel (GSK) combined with SVM. siRNA sequences were represented as vectors in a multidimensional feature space according to the numbers of subsequences in each siRNA and classified into effective or inef-fective siRNAs (14). Ladunga used SVM with polynomial ker-nels and constrained optimization models from 572 sequence, thermodynamic, accessibility, and self-hairpin features over 2200

Table 1.1a Effective and ineffective nucleotides specified in the individual guidelines

Position 1 3 6 10 11 13 16 19

G1 Preferred A T A/C/T A/T

G2 Preferred G/C A/T

Unpreferred A/T G/C

G3 Preferred G/C A T C A/T

Unpreferred T T G

G4 Preferred G/C A/T A/T

G5 Preferred C/G A G T

Unpreferred C A/T G

Position: nucleotide position from 1 to 19 (5 ′ to 3′, cDNA form). Preferred: effective (positive), unpreferred: ineffective (negative).

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书籍介绍

《siRNA和miRNA介导的基因沉默:从实验室到临床应用(导读版)》介绍了RNA干涉成为抑制基因表达和研发治疗试剂的重要方法，然而，这一技术仍然存在诸多问题，譬如传递、稳定性和脱靶作用（非目的基因的沉默和先天免疫系统的激活）的危害。在《siRNA和miRNA介导的基因沉默:从实验室到临床应用(导读版)》中，多位经验丰富的专家探讨了siRNA设计、表达、传递、活体成像、将siRNA不利作用最小化的方法以及在病患中的应用等。作为《分子生物学方法》系列丛书的一卷，《siRNA和miRNA介导的基因沉默:从实验室到临床应用(导读版)》各章针对各自的主题提供了易于使用的最新信息，包括分步骤的实验方案，如新的配方设计和可应用于体内外的实验策略、有效的治疗靶标基因、miRNA功能组分、生源论和病毒感染干涉。