Nucleic Acids and Libraries

Abstract

The invention relates to a nucleic acid comprising the following contiguous elements arranged in the 5 prime to 3 prime direction; a promoter; a selectable marker; a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate miRNA target sequence; and a poly adenylation signal, said elements arranged such that a transcript directed by said promoter comprises said selectable marker, said candidate miRNA target sequence, and said poly adenylation signal in that order. Suitably the miRNA test sequence is or is derived from a 3′UTR. The invention also relates to methods for making and screening libraries.

Description

[0001]The present application is filed pursuant to 35 U.S.C. 371 as a U.S. National Phase application of International Patent Application No. PCT / GB08 / 01176, which was filed Apr. 4, 2008, claiming the benefit of priority to British Patent Application No. GB 0706631.9, which was filed on Apr. 4, 2007. The entire text of the aforementioned applications is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The invention relates to materials such as nucleic acids and libraries for use in functional analysis of regulatory RNAs such as microRNAs (miRNAs), and particularly testing of or screening for targets of regulatory RNAs such as 3′ untranslated region (UTR) sequences.BACKGROUND TO THE INVENTION[0003]MicroRNAs (miRNAs) are now recognized as a novel class of small regulatory RNA molecules that regulate the expression of many genes. They have been shown to mediate angiogenesis, cell adhesion, cell proliferation, survival and play an important role in haematopoi...

AI Technical Summary

Benefits of technology

Therefore, by following the selectable marker or markers, a direct functional readout of the effect of particular miRNAs and those mRNAs can advantageously be obtained. The present invention is based upon this surprising finding. A key advantage of the invention is that is provides a functional readout at the protein level. Although some regulatory RNAs such as siRNA produce cleavage of the target RNA, which allows assay at the RNA level for example by monitoring RNA levels or cleavage, other regulatory RNAs such as miRNAs do not produce this effect. By assaying the effects at the protein level as described herein, numerous regulatory RNA types may be studied functionally, which is an advance compared to prior art techniques.

[0007]c) a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate regulatory RNA target sequence; and

[0008]d) a poly adenylation signal,said elements arranged such that a transcript directed by said promoter comprises said selectable marker, said candidate regulatory RNA target sequence, and said poly adenylation signal in that order.In a first aspect the invention provides a nucleic acid comprising the following contiguous elements arranged in the 5 prime to 3 prime direction;

[0011]c) a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate regulatory RNA target sequence; and

[0013](a) introducing a nucleic acid as described above comprising a candidate miRNA target sequence into a host cell;

Problems solved by technology

Due to the partial homology between a miRNA and its target and inhibition of translation instead of mRNA degradation, target identification is a difficult task.

This is mainly due to the difficulties in identifying targets because, contrary to short interfering RNA (siRNA), miRNA binding is only partially due to homology with the target.

However, the drawbacks of these predictions are that they each generate a substantial number of false positives.

Furthermore, the predictions are likely to be inherently biased as they are mostly based on the knowledge obtained from the very few known miRNA: target interactions, a statistically very small sample size which almost certainly leads to a skew on the predictions.

The prior art study of miRNA gene regulation lacks the necessary tools for target identification and validation, particularly regarding functional studies.

However, since most miRNAs do not have catalytic activity leading to the breakdown of mRNAs, these types of analysis cannot be applied to the study of miRNAs.

However, the drawbacks of these predictions are that they all generate a substantial number of false positives and may be biased as they are mostly based on the knowledge obtained from the few known miRNA:target interactions.

However, computational techniques for finding miRNAs suffer from drawbacks such as being inherently biased towards the small number of miRNAs which have in fact been experimentally verified.

Firstly, this makes it difficult to extrapolate from overlap between the small numbers of known sequences to a wider pool of candidate miRNAs.

Secondly, in any statistically small sample from a large overall group there will be an inherent statistical bias by chance.

Thus, since the number of miRNAs upon which the computational predictions are based is very small, it is almost certain that a strong statistical bias exists in the predictions.

Thus, accurate identification or validation of miRNA / target pairings is a problem in the art.

A key difficulty in the field is the finding of a target for an miRNA.

This is especially difficult since it is known that miRNA targets are not necessarily identical in sequence to the miRNA sequence itself.

Secondly, each individual clone needs to be treated separately since there is no way of separating those harboring nucleic acid of interest from those which do not in a screening type setting.

However, the sensitivity of this technique is very low.

It is very likely that not all proteins are detected by this rather crude methodology.

Clearly, this approach is not sensitive enough for a meaningful study of miRNA action.

Furthermore, as noted above, since miRNAs do not degrade the target RNA in the same manner that siRNAs do, it is also not possible to study miRNA action by monitoring mRNA levels.

Furthermore, there is no functional assay for target discovery in the field of miRNA in existence in the prior art.

In addition, there are examples which expose limitations of the computational models.

Therefore, in addition to predicting targets which are not in fact bound by the miRNA, computational models also do not predict bona fide miRNA pairings.

Therefore, it can be appreciated that these computational systems in the art have numerous serious problems and drawbacks associated with them.

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