Rna array compositions and methods
AssigneeWisconsin Alumni Research Foundation
InventorsLloyd Smith, Cheng-Hsien Wu
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Abstract
Described herein are RNA arrays, and compositions and methods for generating RNA arrays, particularly high density RNA arrays. The disclosed methods for generating RNA arrays utilize template DNA arrays and RNA polymerase to generate RNA arrays. In some embodiments, the disclosed methods use an RNA polymerase and modified ribonucleosides to generate modified RNA arrays for various applications, e.g. RNA arrays having higher nuclease resistance, more conformationally stable RNA arrays, and higher binding affinity RNA aptamer arrays. In some embodiments, the disclosed methods are used to generate RNA bead arrays.
Claims25
An RNA array comprising RNAs that (i) are covalently linked at their 5′ ends to a solid support;
- (ii) represent at least 10 unique RNA sequences;
- and (iii) have a feature density of at least 20 features/cm2.
A template array comprising an array of (i) single-stranded template DNA oligonucleotides linked at their 3′ ends to a solid support, comprising a consensus sequence, and capped by a protecting group at their 5′ end;
- and (ii) single-stranded RNA primers that are covalently linked at their 5′ ends to the solid support, and that are complementary to the consensus sequence, wherein the single-stranded RNA primers hybridize to the single-stranded template DNA oligonucleotides.
A method for generating a template array, comprising
- (i) providing a solid support comprising a layer of protected deoxyribonucleosides that comprise a 5′-photolabile protecting group and are covalently linked at their 3′ end to a spacer layer bound to the solid support;
- (ii) irradiating the layer of protected deoxyribonucleosides with ultraviolet energy sufficient to deprotect about half of the protected deoxyribonucleosides;
- (iii) coupling the deprotected deoxyribonucleosides with a ribonucleoside phosphoramidite comprising a 5′ acid-labile protecting group;
- (iv) irradiating the remaining protected deoxyribonucleosides with ultraviolet irradiation sufficient to deprotect all of the remaining protected deoxyribonucleosides;
- (v) extending the deprotected deoxyribonucleosides, at one or more locations, by light-directed 3′ to 5′ photolithographic synthesis to generate template DNA oligonucleotides of the deprotected deoxyribonucleosides;
- (vi) coupling a protecting group to the 5′ ends of the template DNA oligonucleotides;
- (vii) removing the 5′ acid-labile protecting group on the protected ribonucleosides by acid treatment;
- and
- (viii) extending the deprotected ribonucleosides at one or more locations, by 5′ to 3′ chemical synthesis of RNA primers comprising a sequence that is complementary to a sequence at the 3′ end of the template DNA strands to obtain a template array.
A method for generating an RNA array, comprising
- (i) providing:
- a template array of (a) single-stranded template DNAs linked at their 3′ end to a solid support and comprising a consensus sequence;
- and (b) single-stranded RNA primers that are covalently linked at their 5′ end to the solid support, and that are complementary to the consensus sequence of the single-stranded template DNAs;
- (ii) hybridizing the single-stranded RNA primers with the single-stranded template DNAs;
- (iii) extending the hybridized RNA primers along the single-stranded template DNAs using an RNA polymerase and ribonucleoside triphosphates to obtain double-stranded DNA-RNA hybrids;
- and
- (iv) contacting the DNA-RNA hybrids with a DNase to remove the template DNAs from the DNA-RNA hybrids to obtain an RNA array.
A method to generate an RNA bead pool, comprising:
- (i) providing beads comprising 5′linked RNA primers comprising a consensus sequence;
- (ii) hybridizing the 5′-linked RNA primers with DNA oligonucleotides comprising a unique template sequence and a sequence complementary to the consensus sequence, wherein the DNA oligonucleotides are provided in solution;
- (iii) extending the hybridized RNA primers along the single-stranded template DNAs using an RNA polymerase and ribonucleoside triphosphates to obtain double-stranded DNA-RNA hybrids;
- and
- (iv) contacting the DNA-RNA hybrids with a DNase to remove the template DNAs from the DNA-RNA hybrids to obtain an RNA bead pool.
Dependencies are inferred from the claim text.