Dataset Information

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

ABSTRACT: A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2'-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4?×?10(3) times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume.

SUBMITTER: Wang X

PROVIDER: S-EPMC4272408 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

Wang Xingyu X Li Can C Gao Xiaomeng X Wang Jing J Liang Xingguo X

Molecular therapy. Nucleic acids 20150113

A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2'-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 10(3) times the amount of s ...[more]

PMID: 25584899

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Project description:Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Dataset Information

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

Publications

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

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