Rolling circle amplification of RNA

Abstract

Disclosed are compositions and methods for amplification of RNA molecules. The disclosed method involves synthesizing first strand cDNA molecules from RNA molecules, circularizing the first strand cDNA molecules and replicating the circularized first strand cDNA molecules using rolling circle replication. The method can be aided by the use of specialized primers for cDNA synthesis and specialized probes for circularizing the first strand cDNA molecules. The method can be used to replicate and amplify multiple RNA molecules, such as all RNA molecules in a sample or all mRNA molecules in a sample, or be used to replicate and amplify specific RNA molecules. Rolling circle replication of the circularized first strand cDNA molecules results in long DNA strands containing tandem repeats of the cDNA sequence. The tandem sequence DNA can be used directly (for detection of sequences, for example), further amplified, or used any other purpose. Double-stranded tandem sequence DNA can be used to produce unit lengths of the cDNA sequence. Tandem sequence DNA can also be transcribed to produce transcripts having sequence complementary to or matching the sequence of RNA molecules.

1 . A method of amplifying RNA sequences, the method comprising 
 incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules,    incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules, and    incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication of the circularized first strand cDNA molecules, thereby amplifying RNA sequences, wherein rolling circle replication is primed by a rolling circle replication primer.    
     
     
         2 - 20 . (canceled)  
     
     
         21 . The method of claim  10  wherein the rolling circle replication primer further comprises a promoter portion.  
     
     
         22 . The method of  claim 21  wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.  
     
     
         23 . The method of  claim 22  wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.  
     
     
         24 . The method of  claim 21  wherein each double-stranded tandem sequence DNA comprises multiple tandem repeat units of the same sequence, the method further comprising transcribing the tandem repeat units, whereby tandem repeat transcripts are produced, wherein the tandem repeat transcripts are transcripts of single tandem repeat units.  
     
     
         25 . The method of  claim 24  wherein the tandem repeat transcripts are used, directly or indirectly, in a hybridization assay.  
     
     
         26 . The method of  claim 24  wherein the tandem repeat transcripts are associated with a solid-state substrate.  
     
     
         27 . The method of  claim 24  wherein the tandem repeat transcripts are translated.  
     
     
         28 - 110 . (canceled)  
     
     
         111 . The method of  claim 1  wherein the rolling circle replication primer has a random sequence, wherein the rolling circle replication is primed from a plurality of locations on the circularized first strand cDNA molecules.  
     
     
         112 . The method of  claim 111  wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication of the tandem sequence DNA is primed by the rolling circle replication primer.  
     
     
         113 . The method of  claim 111  wherein the circularized first strand cDNA molecules are amplified via exponential rolling circle amplification.  
     
     
         114 . The method of  claim 1  wherein rolling circle replication is primed by a plurality of rolling circle replication primers, wherein the rolling circle replication is primed from a plurality of locations on the circularized first strand cDNA molecules.  
     
     
         115 . The method of  claim 114  wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication of the tandem sequence DNA is primed by one or more secondary DNA strand displacement primers.  
     
     
         116 . The method of  claim 114  wherein the circularized first strand cDNA molecules are amplified via exponential rolling circle amplification.  
     
     
         117 . The method of  claim 1  wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is not replicated, wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules.  
     
     
         118 . The method of  claim 117  wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.  
     
     
         119 . The method of  claim 117  wherein the tandem sequence DNA is associated with a solid-state substrate.  
     
     
         120 . The method of  claim 1  wherein rolling circle replication is primed by the circularization probe, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is not replicated, wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules.  
     
     
         121 . The method of  claim 120  wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.  
     
     
         122 . The method of  claim 120  wherein the tandem sequence DNA is associated with a solid-state substrate.  
     
     
         123 . The method of  claim 1  wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication of the tandem sequence DNA is primed by one or more secondary DNA strand displacement primers, 
 wherein the rolling circle replication primers have 5′ phosphates, wherein the secondary DNA strand displacement primers have 5′ hydroxyls, wherein the tandem sequence DNA has 5′ phosphates, wherein the secondary tandem sequence DNA has 5′ hydroxyls,    wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the tandem sequence DNA is degraded and the secondary tandem sequence DNA remains.    
     
     
         124 . The method of  claim 123  wherein the phosphate-dependent exonuclease is lambda exonuclease.  
     
     
         125 . The method of  claim 123  wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is used, directly or indirectly, in a hybridization assay.  
     
     
         126 . The method of  claim 123  wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is associated with a solid-state substrate.  
     
     
         127 . The method of  claim 1  wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication of the tandem sequence DNA is primed by one or more secondary DNA strand displacement primers, 
 wherein the rolling circle replication primers have 5′ hydroxyls, wherein the secondary DNA strand displacement primers have 5′ phosphates, wherein the tandem sequence DNA has 5′ hydroxyls, wherein the secondary tandem sequence DNA has 5′ phosphates,    wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the secondary tandem sequence DNA is degraded and the tandem sequence DNA remains.    
     
     
         128 . The method of  claim 127  wherein the phosphate-dependent exonuclease is lambda exonuclease.  
     
     
         129 . The method of  claim 127  wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.  
     
     
         130 . The method of  claim 127  wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is associated with a solid-state substrate.  
     
     
         131 - 138 . (canceled)  
     
     
         139 . The method of  claim 1  wherein the rolling circle replication primer further comprises a promoter portion, wherein the rolling circle replication results in formation of tandem sequence DNA.  
     
     
         140 . The method of  claim 139  wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.  
     
     
         141 . The method of  claim 140  wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.  
     
     
         142 . The method of  claim 139  further comprising transcribing the tandem sequence DNA, whereby tandem sequence transcripts are produced.  
     
     
         143 . The method of  claim 142  wherein the tandem sequence transcripts are used, directly or indirectly, in a hybridization assay.  
     
     
         144 . The method of  claim 142  wherein the tandem sequence transcripts are associated with a solid-state substrate.  
     
     
         145 . The method of  claim 142  wherein the tandem sequence transcripts are translated.  
     
     
         146 - 173 . (canceled)  
     
     
         174 . A method of amplifying RNA sequences, the method comprising 
 incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, wherein the conditions that promote synthesis of first strand cDNA molecules comprise incubating the cDNA primer and the RNA sample in the presence of a reverse transcriptase,    incubating the first strand cDNA molecules in the presence of an RNAse H activity,    incubating the first strand cDNA molecules under alkaline conditions,    neutralizing the first strand cDNA molecules,    purifying the first strand cDNA molecules,    incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules, wherein the conditions that promote circularization of the first strand cDNA molecules comprise incubating the circularization probe and the first strand cDNA molecules in the presence of ligase,    incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication of the circularized first strand cDNA molecules, thereby amplifying RNA sequences, wherein the conditions that promote rolling circle replication of the circularized first cDNA molecules comprise incubating the circularized first strand cDNA molecules in the presence of a DNA polymerase, wherein rolling circle replication is primed by a rolling circle replication primer.    
     
     
         175 . The method of  claim 196  wherein the RNA molecules are mRNA molecules, wherein the cDNA primer is 5′-GTGCGGCCGCTTTTTTTTTTTTTTTTTTTT-3′ (SEQ ID NO:1), wherein the circularization probe is 5′-AAAAGCGGCCGCACNNNNNN-3′ (SEQ ID NO:2), wherein the reverse transcriptase is Superscript II reverse transcriptase, wherein the RNAse H activity is provided by an RNAse H+ reverse transcriptase, wherein the alkaline conditions are produced by adding 0.1 M NaOH, wherein the first strand cDNA molecules are neutralized by adding 0.1 M HCl, wherein the ligase is T4 DNA ligase, wherein the DNA polymerase is φ29 DNA polymerase.  
     
     
         176 . A method of amplifying RNA sequences, the method comprising 
 incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules,    incubating a circularization probe and the first strand cDNA molecules under conditions that promote ligation of the first strand cDNA molecules to each other to form first strand cDNA concatemers, and    incubating the first strand cDNA concatemers under conditions that promote strand displacement replication of the first strand cDNA concatemers, thereby amplifying RNA sequences,    wherein at least one of the first strand cDNA molecules is circularized, wherein the circularized first strand cDNA molecule is replicated by rolling circle replication, wherein rolling circle replication is primed by a rolling circle replication primer.    
     
     
         177 . The method of  claim 176  wherein at least one of the first strand cDNA concatemers is circularized, wherein the circularized first strand cDNA concatemer is replicated by rolling circle replication, wherein rolling circle replication is primed by a rolling circle replication primer.  
     
     
         178 - 179 . (canceled)  
     
     
         180 . The method of  claim 1 , wherein the rolling circle replication primer comprises a complementary portion and a non-complementary portion.  
     
     
         181 . The method of  claim 180 , wherein the non-complementary portion of the rolling circle replication primer is capable of interactions that provide specialized effects.  
     
     
         182 . The method of  claim 181 , wherein the non-complementary portion of the rolling circle replication primer comprises a promoter.  
     
     
         183 . The method of  claim 182 , wherein transcripts are produced from the replicated circularized first strand cDNA molecules, wherein the transcripts are translated.  
     
     
         184 . The method of  claim 183 , wherein proteins are produced by translation of the transcripts.  
     
     
         185 . The method of  claim 181 , wherein the non-complementary portion of the rolling circle replication primer comprises a quencher complement portion.  
     
     
         186 . The method of  claim 1 , wherein the rolling circle replication primer comprises a fluorescent moiety, a fluorescent label, a quenching moiety, or a combination.  
     
     
         187 . The method of  claim 1 , wherein the rolling circle replication primer is a hairpin rolling circle replication primer.  
     
     
         188 . The method of  claim 1 , wherein the rolling circle replication primer includes modified nucleotides.  
     
     
         189 . The method of  claim 1 , wherein the rolling circle replication primer comprises a cleavage site.  
     
     
         190 . The method of  claim 1 , wherein the rolling circle replication primer comprises a primer complement portion.  
     
     
         191 . The method of  claim 1 , wherein the rolling circle replication primer comprises a detection tag.  
     
     
         192 . A method comprising 
 incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, and    incubating a circularization probe and the first strand cDNA molecules under conditions that promote circularization of the first strand cDNA molecules.    
     
     
         193 . The method of  claim 192 , wherein the first strand cDNA molecules are single stranded.  
     
     
         194 . The method of  claim 192 , wherein the 5′ and 3′ terminal nucleotides of the first strand cDNA molecule are base paired to immediately adjacent nucleotides in the circularization probe.  
     
     
         195 . The method of  claim 174 , wherein the RNAse H activity is provided by the reverse transcriptase.  
     
     
         196 . The method of  claim 174 , wherein the RNAse H activity is provided by an enzyme different from the reverse transcriptase.