Properties of Aptamers

The Greek word haptein meaning to attach to is the origin of the word aptamer. Aptamers can be peptides or nucleic acids. Nucleic acid aptamers are single-stranded (ss)DNA or ssRNA molecules that bind to a target with high affinity and specificity, having dissociation constants down to the picomolar range (Jayasena 1999). They typically contain between 25 and 100 nucleotides. DNA or RNA nucleic acid aptamers recognise a large variety of low molecular weight compounds (Gold et al. 1995 Hermann...

RNA Aptamers that Recognize the Cell Surface of Live Trypanosomes

T. brucei was the first protozoan parasite that was targeted using SELEX technology. Homann and Goringer (1999) used African trypanosomes as a model system for the selection of aptamers to the surface of live parasites. The SELEX experiments were designed to identify high-affinity RNA ligands to variant as well as invariant molecules on the parasite surface. Such aptamers might interfere with surface protein function and have the potential to re-direct the immune response to the surface of the...

DNA Vector Mediated siRNAs or Short Hairpin RNAs

SiRNA-directed silencing by transfection is limited in mammals by its transient nature. To overcome some of the shortcomings of transfecting chemically synthesized siRNAs into cells, several groups have developed DNA vector-mediated mechanisms to express substrates that can be converted into siRNAs in vivo (Kennerdell and Carthew 2000 Tavernarakis et al. 2000 Svoboda et al. 2001 Brummelkamp et al. 2002a Lee et al. 2002 McManus et al. 2002 Miyagishi and Taira 2002 Paddison et al. 2002a,b Paul et...

RNAProtein Hybrid Ribozymes

The higher-order structure of mRNAs can interfere with the cleavage reaction, since ribozymes cannot access double-stranded stems within a target RNA. Moreover, an exact folding of the target mRNA and the accessibility to the ribozyme cannot easily be predicted and or determined. Therefore, we postu lated that it would be useful to design a ribozyme that recruits a protein that unwinds interfering secondary structures, thereby rendering the target site more accessible to the ribozyme. To create...

Adenovirus VA RNAs as Suppressor of the RNAi and Interferon Systems

It is well established that most mammalian viruses have evolved defence strategies to suppress the negative effects that the interferon response pathway has on virus multiplication. This is of vital importance for the capacity of a virus to multiply successfully. Numerous viruses encode proteins or decoy RNAs that inhibit the activity of PKR by a surprisingly large range of different strategies (Gale and Katze 1998). For example, human adenovirus type 5 (ad5) encodes two approximately 160-nt...

Expression of Ribozymes In Vivo

The hammerhead ribozyme, named after its secondary structure, which resembles a hammerhead (Fig. 1a), is a small and versatile catalytic RNA molecule that was originally discovered in infectious plant-satellite RNA. The intramolecular cleavage activity of a self-cleaving (cis-acting) hammerhead ribozyme is indispensable for the replication of virusoid molecules. Hammerhead ribozymes have been engineered so that they can cleave any chosen RNA by intermolecular attack in trans (trans-acting)....

Immunotherapy

Oligonucleotides with unmethylated deoxycytidyl-deoxyguanosine (CpG) din-ucleotides have been intensively studied over the past few years as potential immunological adjuvants. CpG-ODNs are able to mimic the immunostimu-latory activity of bacterial DNA, which makes them recognizable by several types of immune cells. As a consequence, they can activate the immune response and lead to the activation of natural killer (NK) cells, dendritic cells, macrophages, and B cells. This characteristic...

References

Agrawal S, Zhang R (1997) Pharmacokinetics of oligonucleotides. Ciba Found Symp 209 60-75 Andreopoulos S (2003) Developing drugs for parasitic diseases. Science 300 430-431 Blum, ML, Down JA, Gurnett AM, Carrington M, Turner MJ, Wiley DC (1993) A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature 362 603-609 Bonora GM, Ivanova E, Zarytova V, Burcovich B, Veronese FM (1997) Synthesis and characterization of high-molecular mass polyethylene glycol-conjugated...

Creation of Allosteric Ribozymes

As mentioned above, ribozymes can target RNAs where a specific sequence is present for efficient cleavage Fig. 2a. The cleavage of a substrate occurs immediately after the NUX sequence (where N is any base and X is A, C, or U) within the target (Shimayama et al. 1995 Zhou and Taira 1998). In some cases, a cleavable triplet sequence is not available at a suitable position within the target. For example, in the case of certain abnormal chimeric mRNAs, which are generated by chromosomal...

Aptamer Core Structures and Serum Stability

The above-described SELEX experiments led to the identification of high affinity RNA ligands that target the trypanosomal surface and thus suggest several pharmacological approaches. In order to optimize the selected RNAs for their pharmaceutical use, the original molecules have to be converted from in vitro binding reagents to therapeutic compounds, which can be tested for their pharmacological effects in vivo. Within this context an important consideration is the cost of synthesis. Since...

Transcriptome Analysis Facilitated by LNA

Efficient selection of polyadenylated mRNA from eukaryotic cells and tissues is an essential step for a wide selection of functional genomics applications, including full-length complementary (c)DNA library construction and sequencing, Northern and dot blot analyses, gene expression profiling by mi-croarrays, and quantitative RT-PCR. The key to successful selection of intact poly(A)+RNA is a fast extraction of total RNA from cells and tissues using strong denaturing agents to disrupt the cells...

Inhibition of HIV Replication by RNAi

Hiv Replication Cycle

The introduction of combination antiretroviral therapy has resulted in a remarkable improvement of the life expectancy of individuals infected with HIV and has significantly reduced their likelihood of developing AIDS. However, despite this progress, HIV infection remains incurable. Toxicity problems associated with current drug therapies and the emergence of drug resistance clearly indicate the need for alternative therapeutic approaches. Retroviral infection with HIV results in the stable...

RNA Targeting by LNAzymes

DNAzymes are catalytically active DNA molecules that are able to cleave RNA in a sequence-specific manner after binding to complementary sequences. The so-called 10-23 DNAzyme Fig. 3 was found by in vitro selection Santoro and Joyce 1997 . It consists of a 15-nt catalytic core flanked by two binding arms that lead to the sequence selectivity of a given DNAzyme. LNAzymes are LNA-modified DNAzymes, and incorporation of two LNA nucleotides in each of the binding arms yielded an LNAzyme with a...

Detection Methods for Small Molecules and Advantages of Aptamers

Small molecules are involved in innumerable biochemical processes in life and can operate as substrate, catalyst or inhibitor in biochemical reactions. Because of these properties, small molecules are valuable as drugs in medicine or in biotechnological applications. In addition, in all cases the feasibility of quickly and reliably detecting them is essential. Different and versatile techniques have been developed for the detection of small molecules. Classic detection methods are based on...

LNA Hybridization Properties and Structural Features

Lna Dna Rna

We have defined LNA as an oligonucleotide containing at least one LNA monomer, i.e., one 2 -0,4 -C-methylene-P-d-ribofuranosyl nucleotide see Fig. 1 Singh et al. 1998 Koshkin et al. 1998b . The LNA monomers adopt N-type sugar puckers, also termed C3 -endo conformations Singh et al. 1998 Obika et al. 1997 . The vast majority of reports on LNA-mediated RNA targeting have dealt with mixmer LNA oligonucleotides, i.e., LNAs containing a limited number of LNA monomers in combination with other types...

SiRNA Agents Work as Ligands for Toll Like Receptors

Surprisingly, recent studies have indicated that siRNAs can induce global up-regulation ofthe expression of IFN-stimulated genes Bridge et al. 2003 Jackson et al. 2003 Sledz et al. 2003 Kariko et al. 2004 Persengiev et al. 2004 Fig. 5 . This effect was detected with synthetic siRNAs that were transfected into cells, and with siRNAs that were produced within cells by the expression of shRNAs. Both of these papers documented significant non-specific changes in gene expression as a consequence of...

Inhibition of HCV by RNAi

HCV is a major cause of chronic hepatitis and hepatocellular carcinoma. Currently, no vaccines are available for HCV, and several groups have used RNAi to target HCV replication. HCV belongs to the family Flaviviridae, and its genome is encoded by a 9.6-kb RNA of positive polarity. Because there is no cell culture system for HCV replication, all studies have used replicon systems in Huh-7 cells as a model for HCV replication. These replicons support HCV RNA transcription and protein synthesis,...

RNA Aptamers that Recognize the Variant Surface Glycoprotein

The aim of the SELEX experiment published by Lorger and co-workers 2003 was to select RNA aptamers that specifically recognize the VSG protein on the surface of bloodstream-stage African trypanosomes. The bloodstream lifecycle stage represents the infectious phase of the parasite, and VSG is the most abundant surface protein during that period. Since the parasite undergoes antigenic variation, which is characterized by the expression of different VSG variants, a selection scheme was designed to...

Nucleic Acid Aptamers Directed Against Carbohydrates

Nucleic acid interactions with polymeric cellular components, particularly with proteins, are dominated by hydrogen bonds, hydrophobic and ionic interactions between phosphate residues and positively charged amino acids. This is defined by the sequence and tertiary structure of the nucleic acid. Carbohydrate residues are void of basic functional groups that are capable of undergoing ionic interaction with nucleic acids. In addition, they possess a very flexible structure. Such molecules are not...

Efficacy and Target Specificity

The biggest difference between ribozyme and siRNA technologies is the recruitment of endogenous proteins applicable in the latter case which is associated with the maintenance of a high intracellular activity. By contrast, the actions of ribozymes do not depend on intracellular factors Scherer and Rossi 2003 . Thus, siRNAs can exploit many intracellular enzymes, such as he-licases and RNases, in cells for cleavage of target mRNAs, and they can be much more effective tools than ribozymes for the...

G V U U Ag

Fig. 1a-c The effects of 2'-amino modification at position 2.1 upon the cleavage activity of a hammerhead ribozyme. a A representative example of multiple turnover reactions of the wild-type ribozyme Wt-URz and the ribozyme with a single 2'-amino group at position 2.1 2'-NH2 2.1 -URz in the presence of 5 mM Mg2 . b The cleavage reaction was performed in the presence of Mn2 . c Summary of the ribozyme catalytic activity following various site-specific modifications at position 2.1. Primary and...

Npcrnas In Vibrio Cholerae

Functional Redundancies and How to Find Them The few examples described here show that the pathogenesis-related RNAs are much more prevalent than previously anticipated. One interesting feature that emerges is the functional redundancy of some of these regulatory RNAs in different pathogenic bacteria Table 2 . It is conceivable that these RNAs are differentially expressed upon different environmental niches conditions or that they have evolved independent regulatory functions i.e., different...