DOCSLIB.ORG

(12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub US 20040O86860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub. Date: May 6, 2004 (54) METHODS OF PRODUCING RNAS OF Publication Classification DEFINED LENGTH AND SEQUENCE (51) Int. Cl." .............................. C12Q 1/68; C12P 19/34 (76) Inventor: Muhammad Sohail, Marston (GB) (52) U.S. Cl. ............................................... 435/6; 435/91.2 Correspondence Address: MINTZ, LEVIN, COHN, FERRIS, GLOWSKY (57) ABSTRACT AND POPEO, PC. ONE FINANCIAL CENTER Methods of making RNA duplexes and single-stranded BOSTON, MA 02111 (US) RNAS of a desired length and Sequence based on cleavage of RNA molecules at a defined position, most preferably (21) Appl. No.: 10/264,748 with the use of deoxyribozymes. Novel deoxyribozymes capable of cleaving RNAS including a leader Sequence at a (22) Filed: Oct. 4, 2002 Site 3' to the leader Sequence are also described. Patent Application Publication May 6, 2004 Sheet 1 of 2 US 2004/0086860 A1 DNA Oligonucleotides T7 Promoter -TN-- OR 2N-2-N-to y Transcription Products GGGCGAAT-N-UU GGGCGAAT-N-UU w N Deoxyribozyme Cleavage - Q GGGCGAAT -------' Racction GGGCGAAT N-- UU N-UU ssRNA products N-UU Anneal ssRNA UU S-2N- UU siRNA product FIGURE 1: Flowchart summarising the procedure for siRNA synthesis. Patent Application Publication May 6, 2004 Sheet 2 of 2 US 2004/0086860 A1 Full-length transcript 3'-digestion product 5'-digestion product (5'GGGCGAATA) A: Production of single-stranded RNA templates by in vitro transcription and digestion With a deoxyribozyme V 2- 2 V 22inv 22 * 2 &3 S/AS - 88.8x, *...* or as IGFR -- is as 4. s Psoi - pursue - - ; B: A western blot showing inhibition of IGF1R in MDA231 breast cancer cells with small Interfering RNAs (siRNAs). 22 and 22inv were chemically synthesised. SIAS and SM/ASM were made with the enzymatic method. 3. Y. e V 3. C: Quantitative analysis of IGF1R inhibition with siRNAs. FIGURE 2 US 2004/0086860 A1 May 6, 2004 METHODS OF PRODUCING RNAS OF DEFINED of an inducible promoter. Although, this method provides a LENGTH AND SEQUENCE Source of continuous production of RNA in the cell, it offers little control over the quantity of the expressed RNA and the FIELD OF THE INVENTION Sequence length. 0001. The invention relate to methods of making RNA 0007. In vitro transcription is relatively cheap and offers duplexes and Single-Stranded RNAS of a desired length and a good approach to Synthesis of large quantities of RNA. Sequence based on cleavage of RNA molecules at a defined Commonly a DNA-dependent RNA polymerase of bacte position, most preferably with the use of deoxyribozymes. riophage origin is used for in vitro transcription. These RNA polymeraseS require a specific promoter Sequence for bind BACKGROUND TO THE INVENTION ing on the template DNA and, for optimal activity, require a downstream Sequence called the “leader Sequence'. The 0002 Small interfering RNAs (siRNAs) are powerful leader Sequence appears at the 5'-end of the in vitro tran laboratory tools for directed post-transcriptional gene Scripts and may be unsuitable in Several applications, Such expression knockdown (Elbashir et al., 2001, Lewis et al., as in siRNA-mediated RNA interference. 2002; Harborth et al., 2001) and inhibition of viral propa gation (Jacque et al., 2002; Gitlin et al., 2002, Jiang and 0008 Donze and Picard (2002) and Yu et al. (2002) both Milner, 2002). The mechanism of action of siRNAs remains describe an in vitro transcription method for production of largely elusive. Data to-date Suggest that SiRNAS may bind siRNAS, which is based on the use of oligonucleotides as to the target mRNA and serve as primers for an RNA templates to produce short transcripts (as first described by dependent RNA polymerase to convert it into dsRNA. An Milligan and Uhlenbeck (1989)). This method is relatively RNase III-type enzyme cleaves dsRNA to produce a pool of Simple and cheap but is limited by Specific Sequence require 21-23 nt or 24-26 nt long dsRNA fragments, thus amplifying ments: all siRNAS made with this method start with a 5'-G the effect of original siRNA. The cellular machinery then residue and require a C-3' residue at position 19 (i.e., uses this new set of siRNAS to repeat the process, Silencing 5'-G-N17-C-3) to allow annealing with the complementary expression of the target gene (Lipardi et al., 2001; Zamore RNA which also has to start with a 5'-G residue due to the et al., 2000; Ketting et al., 2001; Elbashir et al., 2001; requirement by the T7 RNA polymerase. Since it is impor Hamilton A et al., 2002). Specific enzymes involved in tant that the overhang in the antisense Strand is complemen RNAi are largely unknown. However, an evolutionarily tary to the target mRNA, therefore, if the optimal TT-3/UU conserved family of cellular RNase III proteins (named 3' overhang is used in siRNA, the mRNA sequence needs to Dicer enzymes) containing an ATP-dependent helicase-type be 5'-AAG (i.e., mRNA is 5'-AAG-N17-C-3). Efficacy of SiRNAS targeted to different regions of a gene Varies dra domain, two RNase III-type domains and a dsRNA-binding matically. Therefore, these strict Sequence requirements motif appears to be at the heart of RNAi (Filippov et al., greatly reduce the number of potential target Sites for siRNA 2000; Elbashir et al., 2001; Bernstein et al., 2001; Hannon, Selection and are thus disadvantageous in the identification 2002). Genetic studies in Drosophila, Neurospora, Arabi of optimally effective siRNAs. A further disadvantage with dopsis and C. elegans have also revealed Several other this method is that it is not possible to use a leader Sequence candidate genes for potential roles in RNAi. (Williams and in conjunction with the T7 promoter, Since the leader Rubin, 2002; Sharp PA, 2001; Tuschl, 2001; Hannon, Sequence would be transcribed and incorporated into the 2002). siRNA and would ultimately prevent the siRNA from func 0.003 Exogenous siRNA is frequently used in RNAi tioning in RNA interference. Studies. Using chemically Synthesised RNA oligonucle otides, Elbashir et al. (2001) described a systematic analysis 0009. The present invention seeks to provide improved of the optimal Drosophila siRNA duplex. Based on their methods of production of RNAS of defined length and Suggestions, exogenous siRNAS typically consist of a Sequence, particularly siRNAS, by incorporation of an RNA double-Stranded region of 19 base pairs and two nucleotides cleavage Step in order to remove unwanted Sequences. 3' overhangs: -TT-3"/-UU-3' overhangs are preferred over SUMMARY OF THE INVENTION other sequences. For siRNA to be active, it is important that 0010. In a first aspect the invention provides a method of the overhang in the antisense Strand is complementary to the producing an RNA duplex having a defined length and target messenger RNA. Probably, due to Secondary Structure Sequence, the method comprising: in mRNAS, siRNA targeted to different regions of a gene are not equally potent in inhibiting gene expression (Holen et 0011 providing a first primary single-stranded RNA al., 2002; Zhou et al., 2002). Therefore, it may be desirable and cleaving the first primary RNA at a defined to Screen Several SiRNAS to obtain reagents with optimal position to generate a first RNA Strand having a activity. defined length and Sequence, 0012 providing a second RNA strand having a 0004. There are three general methods of producing RNA defined length and Sequence, wherein the first and fragments: chemical Synthesis, intra cellular expression and Second RNA Strands are of complementary Sequence in vitro transcription. These methods have their advantages over at least a portion of their length, and and disadvantages depending on the application. 0013 annealing the first and second RNA strands to 0005 Chemical synthesis of RNAS is relatively straight form an RNA duplex. forward but is expensive. Furthermore, it is difficult to 0014. The invention further provides a method of pro Synthesise chemically RNA fragments that are longer than ducing an RNAStrand having a defined length and Sequence, ~50 nts. comprising: 0006 Intracellular expression requires cloning of a DNA 0015 producing a primary single-stranded RNA fragment into an expression vector, usually under the control including a cleavage Site, wherein the RNA sequence US 2004/0086860 A1 May 6, 2004 upstream of the cleavage Site comprises a leader base-pairs, more preferably from 19 to 22 base-pairs, and Sequence, and the RNA sequence downstream of the most preferably 19 base-pairs. This double-stranded region cleavage Site consists of the defined RNA sequence may be flanked by Short Single-Stranded overhangs. Typi required in the RNA strand, and cally, siRNAS include short (several nucleotide) overhangs at the 3' end, with the 3' overhang in the antisense strand 0016 cleaving the primary RNA at the cleavage site, being complementary to the target mRNA Strand. It is most thereby generating an RNA Strand having the preferred to include 3' dinucleotide overhangs, the most required length and Sequence. preferred being -UU-3'. SiRNAS may also be formed as hairpin RNAS, in which both strands of the siRNA duplex 0.017. The invention still further provides a deoxyri are included within a single RNA strand (Yu, et al. 2002). bozyme or ribozyme comprising a 5" Substrate binding arm, 0024. The methods of the invention rely on cleavage of a catalytic core and a 3' Substrate binding arm, wherein the primary Single-Stranded RNAS at a defined position in order 3' substrate binding arm is capable of specifically hybridis to generate RNA Strands of a required length and Sequence. ing to a leader Sequence present in an RNA molecule under If it is desired to produce an RNA duplex then two such RNA conditions of high Stringency.
Load more

Recommended publications
  • Using Antenna Tile-Assisted Substrate Delivery to Improve Detection Limits of Deoxyribozyme
    University of Central Florida STARS HIM 1990-2015 2015 Using Antenna Tile-Assisted Substrate Delivery to Improve Detection Limits of Deoxyribozyme Amanda J. Cox University of Central Florida Part of the Biochemistry Commons Find similar works at: https://stars.library.ucf.edu/honorstheses1990-2015 University of Central Florida Libraries http://library.ucf.edu This Open Access is brought to you for free and open access by STARS. It has been accepted for inclusion in HIM 1990-2015 by an authorized administrator of STARS. For more information, please contact [email protected]. Recommended Citation Cox, Amanda J., "Using Antenna Tile-Assisted Substrate Delivery to Improve Detection Limits of Deoxyribozyme" (2015). HIM 1990-2015. 1861. https://stars.library.ucf.edu/honorstheses1990-2015/1861 USING ANTENNA TILE-ASSISTED SUBSTRATE DELIVERY TO IMPROVE THE DETECTION LIMITS OF DEOXYRIBOZYME BIOSENSORS by AMANDA J. COX A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Chemistry, Biochemistry Track in the College of Sciences and in the Burnett Honors College at the University of Central Florida Orlando, Florida Fall Term, 2015 Thesis Chair: Dr. Dmitry Kolpashchikov, PhD ABSTRACT One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion – controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit.
    [Show full text]
  • Split Deoxyribozyme Probe for Efficient Detection of Highly Structured RNA Targets
    University of Central Florida STARS Honors Undergraduate Theses UCF Theses and Dissertations 2018 Split Deoxyribozyme Probe For Efficient Detection of Highly Structured RNA Targets Sheila Raquel Solarez University of Central Florida Part of the Biochemistry Commons, and the Biology Commons Find similar works at: https://stars.library.ucf.edu/honorstheses University of Central Florida Libraries http://library.ucf.edu This Open Access is brought to you for free and open access by the UCF Theses and Dissertations at STARS. It has been accepted for inclusion in Honors Undergraduate Theses by an authorized administrator of STARS. For more information, please contact [email protected]. Recommended Citation Solarez, Sheila Raquel, "Split Deoxyribozyme Probe For Efficient Detection of Highly Structured RNA Targets" (2018). Honors Undergraduate Theses. 311. https://stars.library.ucf.edu/honorstheses/311 SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS By SHEILA SOLAREZ A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Biological Sciences in the College of Sciences and the Burnett Honors College at the University of Central Florida Orlando, Florida Spring Term, 2018 Thesis Chair: Yulia Gerasimova, PhD ABSTRACT Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. [1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques.
    [Show full text]
  • Synthetic Biology Applying Engineering to Biology
    Synthetic Biology Applying Engineering to Biology Report of a NEST High-Level Expert Group EUR 21796 PROJECT REPORT Interested in European research? RTD info is our quarterly magazine keeping you in touch with main developments (results, programmes, events, etc). It is available in English, French and German. A free sample copy or free subscription can be obtained from: European Commission Directorate-General for Research Information and Communication Unit B-1049 Brussels Fax : (32-2) 29-58220 E-mail: [email protected] Internet: http://europa.eu.int/comm/research/rtdinfo/index_en.html EUROPEAN COMMISSION Directorate-General for Research Directorate B — Structuring the European Research Area Unit B1 — Anticipation of Scientific and Technological Needs (NEST activity); Basic Research E-mail: [email protected] Contact: Christian Krassnig European Commission Office SDME 01/37 B-1049 Brussels Tel. (32-2) 29-86445 Fax (32-2) 29-93173 E-mail: [email protected] For further information on the NEST activity please refer to the following website: http://www.cordis.lu/nest/home.html EUROPEAN COMMISSION Synthetic Biology Applying Engineering to Biology Report of a NEST High-Level Expert Group NEST - New and Energing Science and Technology - is a research activity under the European Community’s 6th Framework Programme Directorate-General for Research Structuring the European Research Area 2005 Anticipating Scientific and Technological Needs; Basic Research EUR 21796 Europe Direct is a service to help you find answers to your questions about the European Union Freephone number: 00 800 6 7 8 9 10 11 LEGAL NOTICE: Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information.
    [Show full text]
  • Patenting Interfering RNA
    Patenting Interfering RNA J. Douglas Schultz SPE Art Unit 1635 (571) 272-0763 [email protected] Oligonucleotide Inhibitors: Mechanisms of Action RNAi - Mechanism of Action • dsRNA induces sequence-specific degradation of homologous gene transcripts • RISC metabolizes dsRNA to small 21-23- nucleotide siRNAs – RISC contains dsRNase (“Dicer”), ssRNase (Argonaut 2 or Ago2) • RISC utilizes antisense strand as “guide” to find cleavable target siRNA Mechanism of Action miRNA Mechanism of Action Interfering RNA Glossary of Terms • RNAi – RNA interference • dsRNA – double stranded RNA • siRNA – small interfering RNA, double stranded, 21-23 nucleotides • shRNA – short hairpin RNA (doubled stranded by virtue of a ssRNA folding back on itself) • miRNA – micro RNA • RISC – RNA-induced silencing complex – Dicer – RNase endonuclease siRNA miRNA • Exogenously delivered • Endogenously produced • 21-23mer dsRNA • 21-23mer dsRNA • Acts through RISC • Acts through RISC • Induces homologous target • Induces homologous target cleavage cleavage • Perfect sequence match • Imperfect sequence match – Results in target degradation – Results in translation arrest RNAi Patentability issues Sample Claims: • A siRNA that inhibits expression of a nucleic acid encoding protein X. OR • A siRNA comprising a 2’-modification, wherein said modification comprises 2’-fluoro, 2’-O-methyl, or 2’- deoxy. (Note: no target recited) OR • A method of reducing tumor cell growth comprising administering siRNA targeting protein X. RNAi Patentability Issues 35 U.S.C. 101 – Utility • Credible/Specific/Substantial/Well Established. • Used to attempt modulation of gene expression in human diseases • Routinely investigate gene function in a high throughput fashion or to (see Rana RT, Nat. Rev. Mol. Cell Biol. 2007, Vol. 8:23-36).
    [Show full text]
  • Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas Reinhardtii
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of Spring 2013 Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii Xinrong Ma University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Biology Commons, Cellular and Molecular Physiology Commons, Microbiology Commons, and the Molecular Genetics Commons Ma, Xinrong, "Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii" (2013). Dissertations and Theses in Biological Sciences. 51. https://digitalcommons.unl.edu/bioscidiss/51 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII by Xinrong Ma A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences Under the Supervision of Professor Heriberto Cerutti Lincoln, Nebraska May, 2013 SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII Xinrong Ma, Ph.D. University of Nebraska, 2013 Advisor: Heriberto Cerutti RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses.
    [Show full text]
  • Mirna Detection Using a Rolling Circle Amplification and RNA
    biosensors Article MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy Chenxin Fang, Ping Ouyang, Yuxing Yang, Yang Qing, Jialun Han, Wenyan Shang, Yubing Chen and Jie Du * State Key Laboratory of Marine Resource Utilization in South China Sea, College of Materials Science and Engineering, Hainan University, Haikou 570228, China; [email protected] (C.F.); [email protected] (P.O.); [email protected] (Y.Y.); [email protected] (Y.Q.); [email protected] (J.H.); [email protected] (W.S.); [email protected] (Y.C.) * Correspondence: [email protected] Abstract: A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of Citation: Fang, C.; Ouyang, P.; Yang, nucleic acids.
    [Show full text]
  • Retroviral Delivery of Small Interfering RNA Into Primary Cells
    Retroviral delivery of small interfering RNA into primary cells Gregory M. Barton and Ruslan Medzhitov* Section of Immunobiology and The Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520 Communicated by Peter Cresswell, Yale University School of Medicine, New Haven, CT, October 2, 2002 (received for review August 9, 2002) RNA interference is an evolutionarily conserved process in which gene was replaced with the human CD4 gene, which was cloned recognition of double-stranded RNA ultimately leads to posttran- from splenic cDNA. To eliminate any potential signaling, a scriptional suppression of gene expression. This suppression is premature stop codon was introduced after amino acid 425, just mediated by short (21- to 22-nt) small interfering RNAs (siRNAs), after the transmembrane domain. The human H1 promoter was which induce degradation of mRNA based on complementary base cloned from genomic DNA and inserted either upstream of the pairing. The silencing of gene expression by siRNAs is emerging cytomegalovirus (CMV) promoter (RVH1) by using previously rapidly as a powerful method for genetic analysis. Recently, several introduced XhoI and EcoRI sites or within the 3Ј LTR by groups have reported systems designed to express siRNAs in using SalI. The oligonucleotides encoding the human p53 mammalian cells through transfection of either oligonucleotides or siRNA, described by Brummelkamp et al. (6), were 5Ј-GATC- plasmids encoding siRNAs. Because these systems rely on trans- CCCGACTCCAGTGGTAATCTACTTCAAGAGAGTA- fection for delivery, the cell types available for study are restricted GATTACCACTGGAGTCTTTTTGGAAC-3Ј and 5Ј-TCGA- generally to transformed cell lines. Here, we describe a retroviral GTTCCAAAAAGACTCCAGTGGTAATCTACTCTCTTG- system for delivery of siRNA into cells.
    [Show full text]
  • Viral Vectors Applied for Rnai-Based Antiviral Therapy
    viruses Review Viral Vectors Applied for RNAi-Based Antiviral Therapy Kenneth Lundstrom PanTherapeutics, CH1095 Lutry, Switzerland; [email protected] Received: 30 July 2020; Accepted: 21 August 2020; Published: 23 August 2020 Abstract: RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19). Keywords: RNA interference; shRNA; siRNA; miRNA; gene silencing; viral vectors; RNA replicons; COVID-19 1. Introduction Since idoxuridine, the first anti-herpesvirus antiviral drug, reached the market in 1963 more than one hundred antiviral drugs have been formally approved [1]. Despite that, there is a serious need for development of novel, more efficient antiviral therapies, including drugs and vaccines, which has become even more evident all around the world today due to the recent coronavirus pandemic [2].
    [Show full text]
  • Advances in Oligonucleotide Drug Delivery
    REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured
    [Show full text]
  • Ribozyme-Enhanced Single-Stranded Ago2-Processed Interfering RNA Triggers Efficient Gene Silencing with Fewer Off-Target Effects
    ARTICLE Received 9 Dec 2014 | Accepted 21 Aug 2015 | Published 12 Oct 2015 DOI: 10.1038/ncomms9430 OPEN Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects Renfu Shang1,2,3, Fengjuan Zhang1,2,3, Beiying Xu1,2,3, Hairui Xi4, Xue Zhang1,2,3, Weihua Wang1,2,3 & Ligang Wu1,2,3 Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 30 end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 30 overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. 1 National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 2 Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 201204, China.
    [Show full text]
  • Manipulation of Gene Expression in Zebrafish Using Caged Circular Morpholino Oligomers Yuan Wang, Li Wu, Peng Wang, Cong Lv, Zhenjun Yang and Xinjing Tang*
    Published online 22 September 2012 Nucleic Acids Research, 2012, Vol. 40, No. 21 11155–11162 doi:10.1093/nar/gks840 Manipulation of gene expression in zebrafish using caged circular morpholino oligomers Yuan Wang, Li Wu, Peng Wang, Cong Lv, Zhenjun Yang and Xinjing Tang* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38, Xueyuan Road, Beijing 100191, China Received May 18, 2012; Revised August 12, 2012; Accepted August 14, 2012 ABSTRACT strategy with the attachment of multiple caging moieties to the nucleobases or the backbone of phosphate groups. Morpholino oligomers (MOs) have been widely used Ando et al. caged a green fluorescent protein (GFP) to knock down specific genes in zebrafish, but their mRNA and a DNA plasmid through non-specific constitutive activities limit their experimental appli- labeling of the backbone phosphates approximately once cations for studying a gene with multiple functions every 35 bases with coumarin-based photolabile protecting or within a gene network. We report herein a new groups. These caged mRNA and DNA constructs enabled design and synthesis of caged circular MOs (caged the photoregulation of GFP expression in zebrafish and cMOs) with two ends linked by a photocleavable subsequent study of the role of lhx2 in zebrafish forebrain moiety. These caged cMOs were successfully used growth (49,52). More recently, Deiters et al. reported to photomodulate b-catenin-2 and no tail expres- morpholino oligomers (MOs) with multiple photocaged sion in zebrafish embryos. monomeric building blocks and demonstrated the utility of these caged MOs in light-activated control of gene function in zebrafish embryos (26).
    [Show full text]
  • Ribonucleic Acid (RNA)
    AccessScience from McGraw-Hill Education Page 1 of 11 www.accessscience.com Ribonucleic acid (RNA) Contributed by: Michael W. Gray, Ann L. Beyer Publication year: 2014 One of the two major classes of nucleic acid, mainly involved in translating the genetic information carried in deoxyribonucleic acid (DNA) into proteins. Various types of ribonucleic acids (RNAs) [see table] function in protein synthesis: transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) function in the synthesis of all proteins, whereas messenger RNAs (mRNAs) are a diverse set, with each member specifically directing the synthesis of one protein. Messenger RNA is the intermediate in the usual biological pathway of DNA → RNA → protein. However, RNA is a very versatile molecule. Other types of RNA serve other important functions for cells and viruses, such as the involvement of small nuclear RNAs (snRNAs) in mRNA splicing. In some cases, RNA performs functions typically considered DNA-like, such as serving as the genetic material for certain viruses, or roles typically carried out by proteins, such as RNA enzymes or ribozymes. See also: DEOXYRIBONUCLEIC ACID (DNA); NUCLEIC ACID. Structure and synthesis RNA is a linear polymer of four different nucleotides (Fig. 1). Each nucleotide is composed of three parts: a five-carbon sugar known as ribose; a phosphate group; and one of four bases attached to each ribose, that is, adenine (A), cytosine (C), guanine (G), or uracil (U). The combination of base and sugar constitutes a nucleoside. The structure of RNA is basically a repeating chain of ribose and phosphate moieties, with one of the four bases attached to each ribose.
    [Show full text]