Methods for cloning small RNA species

Abstract

This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]The present application claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 60 / 946,922 filed 28 Jun. 2007. The entire teachings of the above application are incorporated herein by reference.FIELD OF THE INVENTION[0002]This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.BACKGROUND OF THE INVENTION[0003]Small, non-coding, regulatory RNA species such as microRNAs have emerged in recent years as a powerful agent in regulating gene expression in eukaryotic cells. First discovered in 1993 (Lee at al., Cell 75: 843-854 (1993)), microRNAs are an abundant new class of regulatory elements that have been shown to impact all aspects of normal cellular processes in both plants and animals, including cell death, differentiation, and proliferation, as well as abnormal processes including cancer (Bartel, Cell 116: 281-297 (2004); Du and Za...

AI Technical Summary

Benefits of technology

[0007]The invention provides improvements to the current methods of small RNA (such as miRNA) cloning to provide greater efficiency and simplification to reduce error-rate. The invention also provides methods to monitor the quality of the reaction using an internal size marker to serve as a quality control reagent.

[0009]The invention additionally provides 5′-5′-adenylated oligonucleotides for ligation of the oligonucleotides using T4 ligase. The adenylated oligonucleotides are made by a novel chemical method by reacting 5′-monophosphate with 5′-silylated phosphate followed by oxidation by N-chlorosuccinimide (NCS) in acetonitrile. Using 5′-monophosphate instead of 5′-pyro- or 5′-polyphosphate eliminates side products because the phosphate backbone is protected by a cyanoethyl group. The adenylation reaction occurs on the support during oligonucleotide synthesis and can be isolated with a single purification. The new method of the invention does not require that adenylation be performed as an additional handling step after oligonucleotide synthesis is complete.

[0010]The invention also provides an efficient alternative to current purification practices. The original protocols for small RNA cloning required three separate denaturing polyacrylamide gel (PAGE) purification steps. Each purification step is time consuming, and each purification results in loss of product. Reducing the number of purification steps improves yield of recovered product. For this reason, small RNA cloning protocols usually require a very large mass of starting RNA. The invention eliminates one of the two PAGE purification steps generally needed to perform cloning via end linkering and therefore improves yield and permits library construction using lower input mass amounts of RNA. The methods also use a reduced amount of ligase when linking the 3′-linker to the RNA product as compared to current methods.

Problems solved by technology

However, the synthetic marker RNA does not contain a 5′-phosphate group which renders it inactive for the 5′-linkering step.

Each purification step is time consuming, and each purification results in loss of product.

For this reason, small RNA cloning protocols usually require a very large mass of starting RNA.

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