A deoxyribozyme that synthesizes 2′,5′-branched RNA with any branch-site nucleotide

RNA molecules with internal 2′,5′-branches are intermediates in RNA splicing, and branched RNAs have recently been proposed as retrotransposition intermediates. A broadly applicable in vitro synthetic route to branched RNA that does not require self-splicing introns or spliceosomes would substantially improve our ability to study biochemical processes that involve branched RNA. We recently described 7S11, a deoxyribozyme that was identified by in vitro selection and has general RNA branch-forming ability. However, an important restriction for 7S11 is that the branch-site RNA nucleotide must be a purine (A or G), because a pyrimidine (U or C) is not tolerated. Here, we describe the compact 6CE8 deoxyribozyme (selected using a 20 nt random region) that synthesizes 2′,5′-branched RNA with any nucleotide at the branch site. The Mn(2+)-dependent branch-forming ligation reaction is between an internal branch-site 2′-hydroxyl nucleophile on one RNA substrate with a 5′-triphosphate on another RNA substrate. The preference for the branch-site nucleotide is U > C ≅ A > G, although all four nucleotides are tolerated with useful ligation rates. Nearly all other nucleotides elsewhere in both RNA substrates allow ligation activity, except that the sequence requirement for the RNA strand with the 5′-triphosphate is 5′-pppGA, with 5′-pppGAR (R = purine) preferred. These characteristics permit 6CE8 to prepare branched RNAs of immediate practical interest, such as the proposed branched intermediate of Ty1 retrotransposition. Because this branched RNA has two strands with identical sequence that emerge from the branch site, we developed strategies to control which of the two strands bind with the deoxyribozyme during the branch-forming reaction. The ability to synthesize the proposed branched RNA of Ty1 retrotransposition will allow us to explore this important biochemical pathway in greater detail

Solid‐Phase Synthesis of Branched Oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143634/1/cpnc0414.pd

Synthesis of Short Oligonucleotides on a Soluble Support by the Phosphoramidite Method

In the last decades, the chemical synthesis of short oligonucleotides has become an important aspect of study due to the discovery of new functions for nucleic acids such as antisense oligonucleotides (ASOs), aptamers, DNAzymes, microRNA (miRNA) and small interfering RNA (siRNA). The applications in modern therapies and fundamental medicine on the treatment of different cancer diseases, viral infections and genetic disorders has established the necessity to develop scalable methods for their cheaper and easier industrial manufacture. While small scale solid-phase oligonucleotide synthesis is the method of choice in the field, various challenges still remain associated with the production of short DNA and RNA-oligomers in very large quantities. On the other hand, solution phase synthesis of oligonucleotides offers a more predictable scaling-up of the synthesis and is amenable to standard industrial manufacture techniques. In the present thesis, various protocols for the synthesis of short DNA and RNA oligomers have been studied on a peracetylated and methylated β-cyclodextrin, and also on a pentaerythritol-derived support. On using the peracetylated and methylated β-cyclodextrin soluble supports, the coupling cycle was simplified by replacement of the typical 5′-O-(4,4′-dimethoxytrityl) protecting group with an acid-labile acetal-protected 5′-O-(1-methoxy-1-methylethyl) group, which upon acid-catalyzed methanolysis released easily removable volatile products. For this reason monomeric building blocks 5′-O-(1-methoxy-1-methylethyl) 3′-(2-cyano-ethyl-N,N-diisopropylphosphoramidite) were synthesized. Alternatively, on using the precipitative pentaerythritol support, novel 2´-O-(2-cyanoethyl)-5´-O-(1-methoxy-1-methylethyl) protected phosphoramidite building blocks for RNA synthesis have been prepared and their applicability by the synthesis of a pentamer was demonstrated. Similarly, a method for the preparation of short RNAs from commercially available 5´-O-(4,4´-dimethoxytrityl)-2´-O-(tert-butyldimethyl-silyl)ribonucleoside 3´-(2-cyanoethyl-N,N-diisopropylphosphoramidite) building blocks has been developedSiirretty Doriast

Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method

The phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143633/1/cpnc0303.pd

Protection of 2′‐Hydroxy Functions of Ribonucleosides

The main purpose of this article is to discuss 2′‐protection in the context of effective oligoribonucleotide synthesis. Emphasis is placed on the 2′‐protecting groups of choice in the synthesis of oligo‐and polyribonucleotides, and the requirements that a protective group must satisfy to become the 2′‐hydroxyl‐protecting group of choice. Finally, the unit discusses the issue of 2′‐O‐acyl and 2′‐O‐silyl group migration to the 3′‐hydroxy function of ribonucleosides during protection, along with the consequences of the conditions used for their removal on the stability of internucleotide linkages.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143728/1/cpnc0202.pd

Branching into RNAi: Synthesis, Characterization and Biology of Branch and Hyperbranch siRNAs

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new medicines from pre-clinical to bedside use. Notably, gene therapy has recently gained widespread traction in cancer research in the advent of the first RNA interference (RNAi) application in humans. RNAi solicits the use of a double-stranded RNA substrate, aptly named short-interfering RNA (siRNA), which binds to and triggers the degradation of a targeted complementary mRNA strand within the catalytic site of the RNA-Induced Silencing Complex (RISC). In this manner, malignant mRNA expression is silenced, thereby inhibiting the translation of proteins that can lead to the production of pathological disorders such as cancer. In spite of their utility, several challenges still remain towards the development of a fruitful cancer-targeting gene therapy approach. Here, a new class of siRNA motifs is presented to increase substrate efficacy in the RNAi application. Our biological target is a member of the heat shock family of chaperone proteins, the Glucose Regulated Protein of 78 kilodaltons (GRP78) which signals tumor initiation, proliferation and resistance towards chemotherapy. Moreover, GRP78 is overexpressed and cell surface localized on a wide range of resilient tumor types but not on healthy cells, making it a viable bio-marker for the development of the proposed cancer-targeting gene therapy approach. Significantly, an efficient solid-phase synthesis method is described for the production of linear, V-shape, Y-branch and hyperbranch GRP78-silencing siRNAs. The novel V-shape, Y-branch and hyperbranch motifs were then studied by CD spectroscopy and thermal denaturation experiments. CD spectroscopy was used to characterize the requisite A-type double-stranded RNA helix for RNAi application; whereas thermal denaturation experiments were used to validate siRNA hybrid stabilities. With stable siRNA hybrids in hand, their biological activity was assessed in HepG2 hepatoblastoma cells, which constitutes a morbid form of pediatric liver cancer and a valid tumor model for studying our GRP78-targeting strategy. The GRP78 silencing activity of the putative branch and hyperbranch siRNAs is discussed and related to its underlying mechanisms for inducing apoptosis in cancer. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. The development of an effective cancer-targeting gene therapy approach is highlighted by preliminary results that showcase the utility of a cancer-targeting peptide (CTP) to condense and deliver siRNA within cancer cells for therapeutic treatment. The latter forms the basis of our cancer-targeting gene therapy approach. Thus, branched and hyperbranched siRNAs may serve as potent siRNA candidates in cancer gene therapy applications

Methods of producing singlet oxygen using compounds having improved functionalization

Novel texaphyrin compounds having improved functionalization are described. Metal complexes of these compounds are active as photosensitizers for the generation of singlet oxygen and thus are potentially useful for treatments performed with singlet oxygen. Several of the metallotexaphyrin complexes absorb light in the physiologically important range of 690-880 nm. The complexes form long-lived triplet states and thus may act as efficient photosensitizers for generation of singlet oxygen.Board of Regents, University of Texas Syste

Introduction to the Synthesis and Purification of Oligonucleotides

Modern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143751/1/cpnca03c.pd

Texaphyrin solid supports and devices

The present invention provides various novel matrix-supported texaphyrins in which a polymeric or solid matrix is covalently modified by the addition of one or more texaphyrins or texaphyrin derivatives. Described are methods of making various polymer-supported texaphyrins, including texaphyrin chromatographic supports, and devices such as catheters, as may be used, for example, in the separation of neutral and anionic species and in applications concerning phosphate ester hydrolysis, other catalytic schemes, MRI, and PDT.Board of Regents, University of Texas Syste