# Patent

## Title
Methods and compositions for discrimination between cytosine and modifications thereof, and for methylome analysis

## Abstract

Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.

## Background
REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/827,885 filed Mar. 14, 2013 which claims priority to U.S. Application No. 61/611,295 filed Mar. 15, 2012. The entire disclosure of each of the following patent applications is hereby incorporated by reference into the present application: U.S. Application No. 61/611,295, filed Mar. 15, 2012; U.S. Application No. 61/722,968, filed Nov. 6, 2012; U.S. Application No. 61/723,427, filed Nov. 7, 2012; U.S. Application No. 61/724,041, filed Nov. 8, 2012; U.S. application Ser. No. 13/804,804, filed Mar. 14, 2013; U.S. application Ser. No. 13/826,395, filed Mar. 14, 2013; and U.S. application Ser. No. 13/827,087, filed Mar. 14, 2013.


BACKGROUND
Cytidine deaminases include activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC). These enzymes are DNA mutators capable of inserting mutations into DNA and RNA by deaminating cytidine to form uridine. These enzymes play a role in antigen-driven antibody diversification processes and in an innate defense system against retroviruses. The human APOBEC family consists of 11 members: APOBEC-1 (Apo1), APOBEC-2 (Apo2), AID, APOBEC-3A, -3B, -3C, -3DE, -3F, -3G, -3H and APOBEC-4 (Apo4). Members of the APOBEC-3A family contain either single (A3A, A3C, A3H) or double (A3B, A3DE, A3F, and A3G) zinc-dependent deaminase domain (ZDD).
Attempts have been made to replace sodium bisulfite methylome sequencing (Frommer, et al., Proceedings of the National Academy of Sciences, 89.5:1827-1831 (1992)) by using AID (Larijani, et al., Molecular Immunology, 42.5:599-604 (2005)). However problems were identified with the use of native AID including: difficulties in obtaining purified active enzyme due to toxicity in non-natural host cells, incomplete conversion of cytosine to uracil arising from low activity of enzymes and substrate bias, and a lack of in vitro assays suitable for selecting AID suitable for methylome sequencing. Hence, methylome sequencing continues to be performed by sodium bisulfite sequencing despite problems associated with this method that include the use of multiple biochemical steps, an inability to distinguish methyl from hydroxymethylcytosine, the requirement for heat to denature the DNA, additional shearing of DNA into small fragments by the chemical treatment, and a limitation on the length of a DNA that can be sequenced.
SUMMARY
In general, in one aspect, a protein variant, for example, a variant of a native cytidine deaminase is provided that includes a protein having at least 90% sequence homology or identity to APOBEC-3A and has at least one mutation. In an embodiment, a protein is provided having at least 90% sequence homology or identity to: (a) SEQ ID NO:1 and comprising at least one mutation at a position corresponding to an amino acid position selected from the group consisting of 25, 45, 109, 123 and 180 of SEQ ID NO:1; or (b) SEQ ID NO:2 and comprising at least one mutation at an amino position corresponding to 23, 25, 29, 45, 69, 104 or 123 of SEQ ID NO:2 or (c) SEQ ID NO:3 (AID) and having at least one mutation corresponding to a deletion at 117 of SEQ ID NO:4.
Embodiments include combining with the protein variant at least one of a purified oxygenase, a polymerase, a polynucleotide and/or at least one primer and dNTPs.
In general, in another aspect, an in vitro mixture is provided that includes a cytidine deaminase and a purified oxygenase.
Embodiments of the mixture may include one or more of the following features: the cytidine deaminase may include AID or mutant thereof; APOBEC or a mutant thereof; APOBEC-3A or a mutant thereof; or the compositions described above; and/or the oxygenase may be a methylpyrimidine oxygenase, for example, mYOX (eg mYOX1) or TET (eg TET1 or TET2 or TET3), a DNA polymerase; and/or at least one primer and dNTPs.
In general in one aspect, an in vitro mixture is provided that includes a cytidine deaminase and a DNA polymerase. The in vitro mixture may further include a polynucleotide and at least one primer and dNTPs.
In general, in one aspect, a method is provided for determining, for a cytidine deaminase, a cytosine preference according to an adjacent nucleotide, which includes: (a) reacting a polynucleotide (optionally single-stranded) containing a cytosine with a cytidine deaminase to convert the cytosine to uracil where the cytosine may be adjacent to any of adenine (A), guanine (G), thymine (T), uracil (U) or cytosine (C); (b) reacting the product of (a) with a glycosylase and an AP endonuclease so as to cleave the polynucleotide at the uracil; and (c) detecting the cleavage product from (b) to determine how the activity of the cytidine deaminase for the cytosine adjacent to any of A, G, T, U or C.
An embodiment may include one or more features for example, the cytidine deaminase may include AID or mutant thereof, APOBEC or a mutant thereof, APOBEC-3A or a mutant thereof or the compositions described above, and/or the polynucleotide may be single-stranded and may be labeled at one end.
In general in one aspect, a method for differentiating an unmethylated cytosine (C) or a 5-methylcytosine (5-mC) from a 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-CaC) or 5-glycosylated hmC (5-ghmC) that includes reacting a polynucleotide optionally containing C, 5-mC, 5-hmC, 5-fC, 5-CaC and/or 5-ghmC, with a cytidine deaminase wherein the 5-mC is converted to a T and only the C is converted to a U; and (b) amplifying or cleaving the polynucleotide to identify the location of at least one converted nucleotide in the polynucleotide.
For aspects of the invention described herein, the cytidine deaminase may be a protein variant as described above; the polynucleotide may be single-stranded, the oxygenase maybe a methylpyrimidine oxygenase such as mYOX1 or a 5-mC oxygenase such as TET1, TET2 and TET3, for example, TET1.
In an embodiment of the method, 5-mC may be differentiated from C, by additionally reacting the polynucleotide prior to (a) with an oxygenase so as to generate a sequence wherein only C is altered to uracil (U).
A further embodiment includes sequencing the polynucleotide in which C is converted to U only and sequencing the polynucleotide obtained in (a) where C is converted to U and 5-mC is converted to T and comparing the sequences to characterize 5-mC in the polynucleotide or alternatively, comparing the sequence of the deaminated polynucleotide from (a) with the sequence of the untreated polynucleotide.
Additional steps may include sequencing a first sample of the polynucleotide after the reaction with the oxygenase and cytidine deaminase to generate a first sequence; sequencing a second sample of the polynucleotide after a reaction with cytidine deaminase but not with the oxygenase to generate a second sequence; and optionally sequencing a third sample of the polynucleotide absent a reaction with the cytidine deaminase or the oxygenase; and comparing the first and at least one of the second and third sample sequences to detect cytosine and 5-mC or comparing the second sample sequence with at least one of the first and third sample sequences.
In another embodiment, cleaving the polynucleotide further includes cleaving the polynucleotide with a glycosylase and endonuclease at a U or cleaving the polynucleotide after DNA amplification, with a restriction endonuclease that recognizes a site after conversion of C to T in the polynucleotide.
Because mYOX1 acts as an oxygenase on single-stranded polynucleotide substrates, cytidine deaminases which also act on single-stranded polynucleotides can be added after the oxygenase reaction without the need to manipulate the substrate and optionally without changing the buffer or reaction vessel. It may be desirable to form the in vitro mixture described above during a polynucleotide analysis for the presence of 5-mC or it may be desirable to form an in vitro mixture of the oxygenase and cytidine deaminase prior to the reaction where the conditions are modulated so that the oxygenase acts on the polynucleotide substrate before the cytidine deaminase acts.
In an embodiment of the method, cytidine deaminase having the characteristics of the protein described above is added to the reaction mixture containing the oxygenase after completion of the oxidation reaction.
In general in one aspect, a method for differentiating a methyl 5-mC from a C is described that includes: reacting a first polynucleotide with sodium bisulfite reaction reagents followed by a cytidine deaminase in the absence of an oxygenase, thereby converting 5-mC to T and converting C to U; and reacting a second polynucleotide, comprising an identical nucleotide sequence, with sodium bisulfite sequencing reagents without subsequent exposure to a cytidine deaminase, thereby converting C to U only.
An embodiment may include amplifying or cleaving the first and second polynucleotides to identify the location of at least one converted nucleotide in the polynucleotides.
An embodiment may include one or of the following features: the cytidine deaminase is APOBEC-3A or variant thereof; the cytidine deaminase is a protein having at least 90% sequence homology with (a) SEQ ID NO:1 and comprising at least one mutation at a position corresponding to an amino acid position selected from the group consisting of 25, 45 109, 123 and 180 of SEQ ID NO:1; (b) SEQ ID NO:2 and comprising at least one mutation at an amino position corresponding to 23, 25, 29, 45, 69, 104 or 123 of SEQ ID NO:2 or (c) SEQ ID NO:3 (AID) and having at least one mutation corresponding to a deletion at 117 of SEQ ID NO:4; the 5-methylcytosine oxygenase is TET1, TET2 or TET3; the methylpyrimidine oxygenase is mYOX1 and/or the polynucleotide has a length of greater than 1 Kb.
An embodiment may include one or more of the following features: adding the cytidine deaminase to the reaction mixture containing the oxygenase after completion of the oxidation reaction; and/or performing the method at a temperature less than 60° C.
The embodiments described above may be used to construct a methylome map.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows that APOBEC-3A expresses well in a cell-free transcription translation system (PURExpress®, New England Biolabs, Ipswich, Mass.) compared with E. coli transformed with DNA encoding APOBEC-3A which did not produce a detectable amount of protein as determined by gel electrophoresis.
Lane 1: Protein ladder (New England Biolabs, Ipswich, Mass.) arrow approximates to a 25 kd band on the gel.
Lanes 2-7: Human APOBEC-3A was subcloned under a T7 phage promoter (2 clones, A3A-1 and A3A-2) and expressed in T7 Express Competent E. coli (New England Biolabs, Ipswich, Mass.). U=uninduced, T=after induction with 0.5 mM IPTG and cultivating 3 hours at 30° C., S=soluble fraction after induction with 0.5 mM IPTG and cultivating 3 hours at 30° C. No distinct bands corresponding to APOBEC-3A were observed.
Lanes 8-9: The same clones (A3A-1 and A3A-2) were expressed using PURExpress; the black arrow points to APOBEC-3A protein.
Lane 10 contains a control sample with no plasmid (A3A-1 or A3A-2) added in PURExpress. No band corresponding to AID or APOBEC-3A was observed.
Lane 11: A gene for human AID was subcloned under a T7 phage promoter, and expressed using PURExpress producing a band having a slightly larger molecular weight compared to APOBEC-3A than expected
FIG. 2A-F shows an assay for determining the conversion of C to U using APOBEC-3A.
FIG. 2A shows 44 bp single-stranded DNA (ssDNA) substrates having an internal unmodified C adjacent to a T and a 3′-FAM marker. The DNA is reacted with APOBEC-3A that converts a C to a U and with USERTM (New England Biolabs, Ipswich, Mass.). USER removes then cleaves at the U thus generating two fragments where the labeled fragment is 26 bp.
FIG. 2B shows a urea gel which reveals the presence of the 26 bp fragment in all samples except the control which was not reacted with A3A and hence only an uncleaved 44 bp band can be seen.
FIGS. 2C-2F show the cleavage pattern for an APOBEC-3A obtained by in vitro transcription-translation in the PureExpress system. This rapid easy assay shows the extent of deamination of APOBEC-3A for AC (FIG. 2C), CC (FIG. 2D), GC (FIG. 2E) and TC (FIG. 2F) substrates at various concentrations.
FIG. 3 shows that primate APOBEC-3A family members vary in target sequence preferences: left to right: Human A3A, Macaca mulatta A3A, Pan troglodytes A3A, and Pan paniscus A3A.
FIG. 4 shows that primate APOBEC-3A family members vary in target sequence preferences: left to right: Pango pygmaeus A3A, Actus trivigatus A3A, Hylobates lar A3A, and Saimiri sciureus A3A.
FIG. 5A-B shows that mutations in human APOBEC-3A polypeptide change target sequence preferences.
FIG. 5A shows mutations in the human APOBEC-3A polypeptide sequences that alter target sequence preferences.
FIG. 5B shows activity of mutant enzymes where the mutation is described on the left and the assay is as described in FIG. 2A.
FIG. 5C shows mutations in Pan troglodytes APOBEC-3A that alter sequence preferences.
FIG. 5D shows the activity of mutant enzymes where the mutations are listed on the left and cleavage is shown for oligonucleotides containing single AC, CC<GC and TC from left to right using the assay shown in FIG. 2A.
FIG. 6A shows the wild type sequence (SEQ ID NO:3) and a mutant sequence (SEQ ID NO:4).
FIG. 6B shows the AID wild type and an AID mutant described in FIG. 6A and the effect of this mutation on altering target cleavage preferences using urea gel electrophoresis and a substrate an oligonucleotide having a single C immediately preceded by an A.
FIGS. 7A and 7B shows an assay for detection of 5-mC in a polynucleotide.
FIG. 7A shows a 71 base ssDNA substrate having an internal TmCTAA that is reacted with APOBEC-3A. Conversion of mC to T by APOBEC-3A and subsequent PCR amplification results in conversion of the sequence to a double-stranded product of PCR containing a TTTAA sequence which can be cleaved by the restriction endonuclease Msel (New England Biolabs, Ipswich, Mass.) (which recognizes TTAA) into two fragments, a 39 base pair fragment and a 77 base pair fragment. The bands can be readily identified using gel electrophoresis.
FIG. 7B shows a gel demonstrating the presence of cleavage products in the form of two reduced molecular weight bands on the gel.
FIG. 7C shows that the ability of a deaminase to convert 5m to T is unaltered in the presence of an adjacent U where the U might arise after conversion from a C to U by a bisulfite sequencing reaction.
FIG. 7D shows a two-fold serial dilution of APOBEC-3A on the reaction described in FIG. 7C. Lanes 3-9 show 77 bp and 39 bp fragments and no 116 bp DNA demonstrating complete conversion of 5-mC to T.
FIGS. 8A and 8B shows that 5-hmC is recognized as a C in the presence of APOBEC-3A.
FIG. 8A provides the labeled single-stranded polynucleotide synthesized with a 5-hmC or hydroxymethyl uracil (5-hmU).
FIG. 8B is a gel which shows that in the presence of USER, only the control polynucleotide containing 5-hmU was cleaved into two fragments. The polynucleotide with 5-hmC remained intact for all enzyme concentrations tested.
FIG. 9A-B shows prior art methods for identifying 5-mC and 5-hmC residues in a substrate DNA.
FIG. 9A shows a method called OxBS-Seq (Booth, et al. Science, 336.6083:934-937 (2012)). This method requires two bisulfite sequencing steps. In the first sequencing step, 5-hmC in genomic DNA is oxidized to formylcytosine (5-fC) by KRuO4, a reagent that is destructive for DNA. 5-fC is subsequently converted into T by sodium bisulfite treatment. In the second bisulfite sequencing step, genomic DNA is subjected to bisulfite treatment without KRuO4 treatment. The first sequencing step reveals sites of 5-mC; this information is subtracted from the 5-mC plus 5-hmC sites provided by the second traditional bisulfite sequencing step.
FIG. 9B shows a method called TAB-Seq (Yu, et al., Cell, 149:1368-1380 (2012)). This method can distinguish 5-hmC from 5-mC after one bisulfite sequencing step. However, multiple sequential enzyme reactions are required. 5-hmC is selectively protected from TET-mediated oxidation and bisulfite conversion by βGT-catalyzed glucosylation of 5-hmC to glycosylated 5-hmC. Next, 5-mC is oxidized by TET to 5-caC, and subsequently converted into T after bisulfite treatment and PCR. Therefore, TAB-Seq reveals sites of 5-hmC in genomic DNA.
FIG. 10A-C shows an overview of locus specific differentiation of 5-mCs from 5-hmCs in bisulfite converted DNA.
FIG. 10A shows a substrate with C, 5-mC and 5-hmC. Sodium bisulfite sequencing converts the C into U but does not affect 5-mC or 5-hmC.
FIG. 10B shows the same initial substrate as in FIG. 10A except the sample is deaminated using APOBEC-3A or AID so that the 5-mC is converted to a T, the C is converted to a U and the 5-hmC is unchanged.
FIG. 10C shows the same initial substrate as in FIG. 10A but this time, oxidation of the 5-mC and 5-hmC is achieved using TET1 or a methyl pyrimidine oxygenase (mYOX1) to convert the 5-mC and 5-hmC to 5-caC. Sequencing identifies these bases as C.
FIG. 11 show a method to detect 5-hmC and 5-mC in a two-step method that first utilizes sodium bisulfite after which the sample is split into two aliquots where one aliquot is not further treated while the other aliquot is deaminated. A comparison of sequences reveals that those Cs in the non-deaminated aliquot that are absent in the deaminated aliquot are 5-mC. Because enzymatic deamination achieves the conversion of C to U, the sodium bisulfite step is optional if it is followed by an enzymatic deamination step.
FIG. 12A-D shows examples of sequences and their fate as they are utilized in the method described in FIG. 10.
FIG. 12A shows a 389 bp substrate in which the methyl CpG are underlined.
FIG. 12B shows a 389 bp substrate where after sodium bisulfite conversion, all Cs converted to Us except methyl in CpG sites.
FIG. 12C shows a 389 bp substrate where after treating with APOBEC-3A enzyme. 5-mCs at CpG sites were converted to Ts.
FIG. 12D shows that after PCR amplification all Us become Ts.
FIG. 13 shows a method for detecting C, 5-mC and 5-hmC in the absence of a bisulfite sequencing step. An oligonucleotide is either reacted with an oxygenase (for example, TET1 or mYOXI) and a deaminase (APOBEC-3A or AID) or with a deaminase alone and then amplified and sequenced. APOBEC-3A converts a C to U but 5-mC and 5-hmC are oxidized to 5-caC which will be identified as C during sequencing. Thus the combination of APOBEC-3A or AID with an oxygenase has the same effect as sodium bisulfite sequencing. If the same oligonucleotide is treated with APOBEC-3A or an AID only then 5-mC will be converted to a T. If the amount of C and 5-mC is known than the sum total of 5-hmC+5-fC+5-caC can be calculated.



DETAILED DESCRIPTION OF THE EMBODIMENTS
The term “cytidine deaminase” is intended to encompass a naturally occurring enzyme, or an enzyme which is not known to be naturally occurring having at least 85%, 90%, 95% and 99% sequence similarity or identity to the wild type enzyme, where the enzyme may be mutated, the mutations optionally including one or more artificially introduced point mutations or deletions.
A wide range of cytidine deaminases have been identified from humans and other species by means of sequence homology. Eleven members of the human APOBEC-3A family are listed in Table 1. The table also lists preferences in recognition of nucleotide motifs, DNA substrate preference for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and biological functions of the enzymes. The cytidine deaminase family of proteins can be identified in amino acid similarity searches through the occurrence of the ZDD (H/C)-x-E-x25-30P-C-x-x-C. The sequence for human APOBEC-3A is provided in FIG. 5 (SEQ ID NO:1) and human AID is provided in FIG. 6 (SEQ ID NO:3).
The term “oxygenase” includes enzymes that catalyze the conversion of 5-mC and 5-hmC to 5-fC to 5-caC. The family that includes mYOXI is variously referred to as methylpyrimidine oxygenase, a cytosine oxygenase, and a 5-methyl oxygenase. Examples of mYOX include the following:















SEQ





ID



Name
Accession #
NO:
SEQUENCE







mYOX1
XP_002667965.1
24
MTTFKQQTIKEKETKRKYCIKGTTANLTQTHPNGPVCVNR





GEEVANTTTLLDSGGGINKKSLLQNLLSKCKTTFQQSFTN





ANITLKDEKWLKNVRTAYFVCDHDGSVELAYLPNVLPKEL





VEEFTEKFESIQTGRKKDTGYSGILDNSMPFNYVTADLSQ





ELGQYLSEIVNPQINYYISKLLTCVSSRTINYLVSLNDSY





YALNNCLYPSTAFNSLKPSNDGHRIRKPHKDNLDITPSSL





FYFGNFQNTEGYLELTDKNCKVFVQPGDVLFFKGNEYKHV





VANITSGWRIGLVYFAHKGSKTKPYYEDTQKNSLKIHKET





K


 


mYOX6
XP_002674105.1
25
MPMNYITSDLKTQLGEYLIGIVNPMLDETITAALEILSPR





TINYLTSLPHPYHILNNCIYPSTAFNYLEPQIEKHRIKNA





HKDTRDATPSVLFYLGDYDEKEGYLEFPEQNCKVFVKPGD





LLLFKGNKYKHQVAPITSGTRLGLVYFAHKACKVMDFYDD





YQKESLNKHKQQNQ


 


mYOX4
XP_002676528.1
26
MSINTTFNQKTTQSGEPPMMMRMTNSSTPPLTPKNCLPIF





VYNDYGKLIREEQQQPTDIITNNNNSMMRSMPTTNRWETN





PQTPLSVSPFQPLLPIPNFSHAFIVGNLPPSVSVRRKNRK





MSEKPKNNSAPSKIMHQLELSVLNNQRRIAPKGPLADISN





IQLPQQESTNKSNNTTPKKPRIRQLMLTTPLRESLQSNQS





ARSKYIDEEANNYSINDSPETTIIKTSNTKDSEHKAAMAT





NLGLSTDDFECKPFETTTLPSVIDKNYLVVDKEGCTQLAL





LPNHIPTSVCKLIEVKCRKVSNLRHALKIQKASFYVNWWT





KSQPMGYMCKDNESEIGKVVNEIAELLSDHCRNLLRMCNE





RVYKKISELKEDKFFAPCICFNILEHDLESRITKFHHDKM





DYGVSVLFYFGDYSRGNLNVLDAGSSSTIVTRPGDAVILR





GNYYKHSVCINIEPGNNKARYSIVFFAHSTHFLKKKYELS





PAAAKKAFLVDNPDFVSIKKRKQASSSSDVSVKKSKKSTE





DNVEFIQTHTYLGNGYKSGHKNYQYYVKFNNSDQKEWKSY





ESLPKQAVASYWVKFKKLKSLSNQ


 


mYOX7
XP_002668594.1
27
MLEAQHHKLTIYTGMWGHMKPCVFIAADNCNKSGETIVEN





LLFKLGKIGSKLMEILSPFTMNFLSSLDPEIFLNHDLFPI





SATNFMIPGNKHRILKPHKDNQDVGLCIIFYFGNYNAPLE





FVNKGSVFNTERGDVLLMRGSHFRHVVKPVDNGLLEHVHD





PMRISVVLFAHKSLKMNPSYFLNAGSALKAHDEDFPEKAK





KRKKKRK


 


mYOX8
XP_002676954.1
28
MFLRNILPENTTTEVTNILDKINQRRSKENYYIGSWGKSS





SFLFKTNDTIFNELSSQFIKIINLLKNYVLEILKFGNNKM





RKFLEKYNSSDFLSIYPTVCFNFLDKSVDENRILHIHPDK





EDTGTSLIFYFGKFKGGAISFPELNFKLMVQSADVLLFDG





KNNLHAVESLHGKDDVRYSVVFFAHKADLGKTSYPMNRGE





VMKGIKNKINN


 


mYOX5
XP_002668409.1
29
MDIGIDWRGTHFRHKNHLVKEEVCDRTNWIVLCPNGQVDI





AFFPNAIPEELCLEMETVVANSDVDILSCKKAIIDGSWTR





YGNGIYPVKTITTNQSILLHELNDKCGPFVLDKLKHINKN





MFNKLDNINEDIKNYKIFAKYPTLALNVSHNENYNISKKP





YRKHTDGNDIGLGVLTYFGSEIIEGGNLIIHIENLKVFNF





PIQRRDLVFLNSKFYAHQVTKVTSGIRFGLVYFAGEAHFR





VRNNDDFLPALPFNANDKELREERSKKGRKSMNEYKKRFL





KKYLREKKKINKKRVKCKNKLK


 


mYOX2
XP_002682154.1
30
MGPLHVSQHDKKKPKHRRRKKQFLKAQALTRVCWENEKSI





DESGKTRVYKMIKEWEFLKGNNIQSNEPILSVYGVNDTIP





KEISSNTIIVTKEGMVEMALLKSVLPPSLLEECTQLCREM





SEWLATEKDIDKGSFFSGWWTMNMPMGYKCADSFRFELVD





TKVKQIQALLHDTFQHILELANPKLFAKLSKLTERGQTPV





VCFNMIPTRNESVKEKFQGSYKSTDKVNRPKTNHRDRNDM





GISAMFYMGKFGGGSLQLIRVNEHTPKTLVHIQAGDVVLL





RANKYRHAVSPTRPQSFPLANSSQTEVDDVKICENSSPTL





NNPQADDNTPTLINTCPKQEPTDGDNPVQSSKEPSNDYEQ





KRFSFIFFAHRSHFKHSKVYCGMGQRQALNAFKADHPYYQ





SQRMKKKLGDDCLDQSLILTEKRKPIKRNYALFNECGDDK





QEESDEEEYQQYEPKPTTEEYTIKVIVDHEKVFKGSDQSR





KSYLYHIQWLGYPDETWEPYEHLDDCQVFEDYLKHHNISL





FDEEEEDRKVDDSMLLPAWMHEDESLFEALLPIICCSTDN





PRHHLDDVPPFDFNY


 


mYOX3
XP_002668005.1
31
MTEIVELSNIEPKDQKQAIIGGTWNRYGNSIEIVAGISDE





NNTLLDNLTNCCESFVLDKLWHLNRSMYNKLDTIEEKIKN





FKTYAKYPSLALNLLCKENYNGKVKPYRKHIDPNNNGMDV





LMFFGKTFEGGNLIVSYHYTNIDFRMFTLPIQSGDLVFLN





SRIYHHKVTKVTSGVRCGLVFFAGLDHFSVRKANYKKVKK





EEYQKNMDDKLLALPFQQKDKDLRIERTKTGRKEIKQFHK





NLQNNLPNKKRKK









The term “polynucleotide” includes a DNA or RNA having ranging in size from a few nucleotides (for example, 10 nucleotides) to a genome length. A polynucleotide as used herein may also be 1 kb or 2 kb or 5 kb or 10 kb in length unless otherwise specified.
The term “DNA polymerase” includes any polymerase suitable for amplifying DNA by isothermal or temperature cycling amplification methods.
In general “detecting”, “determining” and “comparing” refer to standard gel based techniques such as SDS gels or TBE-urea gels described in the examples and equivalent methods well known in the art. These terms may be applied to sequencing, where DNA sequences are compared. There are a number of sequencing platforms that are commercially available and any of these may be used to determine or compare the sequences of polynucleotides.
The term “sodium bisulfite sequencing reagents” refers to a standard method for detecting 5-mC as is described in Frommer, et al., Proceedings of the National Academy of Sciences, 89.5:1827-1831 (1992).
A number of problems had to be solved to achieve the present embodiments. These include:

    
    
        (a) a means to generate sufficient quantities of purified APOBEC-3A/AID to test reproducibly for deamination specificity;
        (b) a simple, reliable assay was developed to determine whether the activity and specificity of an cytidine deaminase or its homolog or derivative might be suitable for the desired purpose; and
        (c) determination of whether and to what extent any purified cytidine deaminase obtained from a natural source could deaminate a C adjacent to an A, T, G or C.

Means for Generating Sufficient Quantities of Purified APOBEC-3A

    
    


An in vitro transcription and translation system was successfully utilized to generate purified active APOBEC-3A and AID from a synthetic gene sequence. The product of synthesis could then be tested on synthetic oligonucleotide substrates containing a modified C in varying sequence contexts. The sequences of the oligonucleotides can be essentially any sequence containing a C or modified C although in embodiments of the assays, the oligonucleotide preferably has a single C unless otherwise specified with an alternative base immediately preceding the C. FIGS. 8-11 show that APOBEC-3A and AID express well in in vitro transcription translations systems.
An Assay for Cytidine Deaminases
Synthetic oligonucleotide substrates were prepared which contain a single internal C and a 3′ label (Integrated DNA Technologies, Coralville, Iowa). A synthetic oligonucleotide with multiple internal Cs may also be used as a substrate, to generate multiple fragments in the assay described in FIG. 2. A synthetic oligonucleotide can be substituted by a naturally occurring DNA fragment which is denatured so as to provide single-stranded fragments suitable for reacting with APOBEC-3A and AID. Using substrates designed as described above, assays were designed to: (a) analyze the specificity of an APOBEC-3A or AID or variant thereof; (b) differentiate C from 5-mC; and (c) differentiate 5-hmC from 5-mC.
(a) Use of a cleavage agent for breaking ssDNA into 2 fragments at a cytidine deaminase modified nucleotide.
This assay for determining the activity of a cytidine deaminase relies on a change in a ssDNA that results in selective cleavage using a reagent. Although the detectable marker illustrated in FIG. 2A and FIG. 2B and in Example 2 was a fluorescent label, any label known in the art capable of attaching to the 3′ end or the 5′ end of an oligonucleotide and being detectable on a gel or other separation device, can be used. Examples of detection labels and capture tags for oligonucleotides are described in U.S. Pat. No. 6,368,801.
The conversion by APOBEC-3A or AID of a C into a U can be detected rapidly and simply by reacting the oligonucleotide with a glycosylase/endonuclease mixture such as the commercially available USER or an equivalent which removes the U from the DNA thereby generating two fragments from the oligonucleotide, one of which retains the fluorescent label. Since the labeled cleavage product is significantly smaller than the full-length oligonucleotide, it can readily be detected by size separation such as shown using gel electrophoresis. FIGS. 3 and 4 show assay results for 8 human and simian APOBEC-3A enzymes using the rapid assay shown in FIG. 2 on PURExpress generated samples. An advantage of this assay and others that utilize cytidine deaminase is that the temperature of the reaction may be less than 60° C. for example, less than 50° C., or less than 40° C.
(b) Restriction endonuclease cleavage of dsDNA only after cytidine deaminase induced modification of a C to a T.
A change of a C to a T in a reaction with APOBEC-3A or AID was designed to result in the formation of a specific restriction endonuclease cleavage site that was not present previously. When this modified oligonucleotide was amplified, the resulting dsDNA could be cleaved by the restriction endonuclease to generate two fragments where previously only one was present. Example 3 and FIG. 7A-D describe one instance of this method. PCR amplification is described here though any form of amplification can be used including various isothermal amplification methods such as transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification.
(c) Conversion of 5-hmC to 5-hmU.
The oligonucleotides were reacted with a cytidine deaminase so that the 5-hmC was converted to 5-hmU. 5-hmU could be cleaved with a glycosylase and an endonuclease. In the example, SMUG (New England Biolabs, Ipswich, Mass.) was used with EndoVIII (New England Biolabs, Ipswich, Mass.) to cleave the synthetic oligonucleotide. A cleavage fragment could be detected by a label either at the 3′ end or the 5′ end of the oligonucleotide although a same, similar or different label could be used at either end of the oligonucleotide substrate to facilitate detection of the cleavage product. Example 4 and FIG. 8 show results obtained using a 3′FAM label on a synthetic oligonucleotide with a single internal 5-hmC located in a sequence TCT in a substrate oligonucleotide and treated with APOBEC-3A.
The ability to readily make protein variants and the robust assay both described herein, provides a simple means to generate and evaluate mutants of cytidine deaminases for improved catalytic conversion of C and 5-mC in polynucleotide substrates that may be ssDNA fragments or genomes. An advantage of the methods described herein are that they are suited for analyzing large pieces or even entire genomes without the difficulties of shearing that arise when a temperature or alkali denaturation step is required or where the chemical also cleaves the DNA, as in bisulfite sequencing.
Mutants of APOBEC-3A and AID were generated that had enzymes with reduced or no bias to any particular sequence context for converting a C to U or a 5-mC to T. For example, using a human APOBEC-3A sequence as a starting point, mutations were introduced into the gene and the mutants tested in the assay described above. The results for 12 mutants of two wild type APOBEC-3A are shown in FIGS. 5A-D and for 1 mutant of AID in FIG. 6A-B. These mutations are intended to be representative and are not intended to be comprehensive. It would be straightforward based on the compositions and methods described herein to select a sequence of a cytidine deaminase such as APOBEC-3A from a sequence database, introduce mutated amino acids into the protein sequence and assay for altered substrate specificity using embodiments of the methods described here.
Various combinations of the 5 mutants of human APOBEC-3A and 7 mutants of Pan troglodytes APOBEC-3A are shown in the examples and in FIG. 5A-D that have altered cleavage bias of substrates. For example, for human APOBEC-3A, E109 could be combined with one or more of any of G25, S45, R123 and D180, for example E109A could be combined with one or more of any of G25V, S45W, R123H and D180E. Similarly, R123 could be combined with one or more of any of E109 G25, S45, and D180 for example, R123H could be combined with one or more of any of E109Q G25V, S45W, and D180E. Similarly, D180 could be combined with one or more of any of G25, S45, R123 and E109 for example D180E could be combined with one or more of any of G25V, S45W, R123H and E109Q. Similarly, S45 could be combined with one or more of any of G25, E109, R123 and D180 for example S45W could be combined with one or more of any of G25V, E109Q, R123H and D180E. Similarly G25 could be combined with one or more of any of E109, S45, R123 and D180 for example G25V could be combined with one or more of any of E109Q, S45W, R123H and D180E. In one example, human APOBEC-3A with an E190 mutation for example E109Q was used in embodiments of the method. In another example human APOBEC-3A with G25, S45, E109, R123 and D180 mutations for example G25V, S45W, E109Q, R123H and D180E mutations were used.
For Pan troglodytes APOBEC-3A, one or more of mutations at positions corresponding to 23, 25, 29, 45, 69, 104 and 123 can be introduced into (SEQ ID NO:2) to alter the sequence preference for the enzyme. Examples of specific mutations correspond to 23N, 25V, 29H, 45W, 69R, 104W and 123H. Examples of combinations of mutations include combining a mutation at positions corresponding to 23 with one or more or two or more mutations at positions corresponding to 25, 29, 45, 69, 104 and 123; or 25 with 29, 45, 69 104 and 123, or 29 with 45, 69, 104 or 123, or 45 with 69, 104 or 123, or 69 with 104 or 123, or 104 with 123. Three or more or four mutations selected from positions corresponding to 23, 25, 29, 45, 69, 104 and 123 can be selected or a mutant may be constructed that includes 5 mutations at positions corresponding to 23, 25, 29, 45, 69, 104 and 123 for example 23N, 25V, 29H, 45W, 69R, 104W and 123H.
With the disclosed assay, it is possible to mutate any cytidine deaminase at any site and test the mutant for altered site preference. In one embodiment of the invention, a cytidine deaminase with a site preference is selected to determine the locations of a subset of 5-mC residues present in a target nucleic acid.
In another embodiment of the invention, a cytidine deaminase with little or no site preference is preferred. Accordingly, a mutated APOBEC-3A having a mutation corresponding to E109Q or S45W from SEQ ID NO:1 or C69R, T23N, or G25V in SEQ ID NO:2 may be selected with these features.
Mutants of wild type AID (SEQ ID NO:3) were created and can be used in embodiments of the methods described herein. For example a mutation at position 117 may be introduced, for example a deletion as shown in FIG. 6A (SEQ ID NO:4).
In one embodiment, a method is provided which shows a time course leading to complete conversion to U of all 5-mCs in a substrate containing multiple 5-mCs which when amplified resulted in a T in place of a U (see for example FIG. 8A-8B). APOBEC-3A is demonstrated to convert all of the 5-mC into U in a time that is greater than 2 hours although even after 1 hour about 95% is converted. It is expected that manipulating conditions such as one or more of pH, concentration and buffer and selected APOBEC-3A or AID variants results in substantially 100% conversion in a time frame of less than 2 hours.
Methylome Sequencing
Sodium bisulfite sequencing has become an established method for mapping 5-mC in a genome as part of an epigenetic study. Unfortunately, sodium bisulfite sequencing cannot differentiate between 5-mC and intermediates of demethylation such as 5-hmC, 5-fC and 5-caC. In brain tissue, there is a significant amount of 5-hmC as well as small amounts of 5-fC and 5-caC while in all tissues, there are at least some of these modified bases. Another problem associated with sodium bisulfite sequencing is that the method damages the target DNA causing extensive fragmentation. This complicates assembly of maps for a methylome. Another problem of sodium bisulfite sequencing is that it involves multiple chemical steps and therefore is intrinsically inefficient and costly to perform. Nonetheless, an embodiment of a method is provided that facilitates sodium bisulfite sequencing and ameliorates one or more of the above limitations. Accordingly, a one-enzyme step enables mC to be differentiated from a 5-hmC (see FIG. 11). The sodium bisulfite reaction which precedes the deamination reaction was shown not to interfere with this reaction (see FIG. 11).
Embodiments of the invention include methods for methylome construction that may utilize sodium bisulfite sequencing while reducing the number of steps to determine not just the occurrence of modified bases but the occurrence of methyl bases and not hydroxymethyl bases. Other embodiments do not utilize sodium bisulfite sequencing at all but rather utilize two enzyme reactions, in particular, a demethylase such as methyl-pyrimidine oxygenase or TET or analog thereof and an AID. A comparison between these oxygenases is given below. An example of the class of enzymes referred to as methyl pyrimidine oxygenases is an enzyme identified as mYOXI which is described in U.S. application Ser. No. 13/827,087.







TABLE 1





Properties of oxygenases

















Name
Methyl-pyrimidine oxygenase
5-methylcytosine oxygenase (TET)


Length
~300AA
~1600AA


Reaction temp.
34 C.
37 C.


Cofactors
2-oxoglutarate and Fe2+
2-oxoglutarate and Fe2+


Optimal pH
6-6.5
8


Substrate
DS-DNA, SS-DNA
DS-DNA


forms


Substrate
5-mC (and depending on enzyme, T)
5-mC


Products
5-mC->5-hmC/5-fC/5-caC, T->5-
5-mC->5-hmC/5-fC/5-caC



hmU/5fU/5-caU


Substrate
Converts mCG to >90% 5-caC, coverts
Similar to mYOX1


specificity
mCWG to a mix of 5-hmC/5-fC/5-caC


ATP effect
Inhibition
stimulation


Conserved
Contains characteristic 2OGFE-domains,
In addition to 2OGFE-domains,


Sequence
presumably for binding 2OG and Fe2+
there are long extra sequences.


feature









FIG. 10A-C shows a comparison of: sodium bisulfite sequencing in which an unmodified C is deaminated to form a U leaving 5-mCs detected as a C (FIG. 10A); enzymatic deamination only (FIG. 10B) in which an unmethylated C is converted to U, a 5-mC is converted to a T and no change is observed with 5-hmC which is read as a C in the sequencing reaction; and with an oxygenase reaction step (FIG. 10C) in which a 5-mC and 5-hmC are converted to 5-caC which is recognized as C and thereby replicates the sodium bisulfite sequencing reaction. When parallel reactions are analyzed for samples+/−enzymatic deamination, after sodium bisulfite sequencing step, the results reveal which C residues were methyl and which were hydroxymethyl by subtraction (FIG. 11).
The reaction pathways in FIG. 13 remove the need for a sodium bisulfite sequencing reaction. This method contrasts with TAB-seq (Yu, et al. (2012)) which requires two separate enzyme reactions in addition to sodium bisulfite sequencing. In TAB-seq, 5-hmC is first labeled with a glucosyl transferase, and 5-mC is oxidized prior to sodium bisulfite sequencing to 5-caC which is then converted to 5-caU and hence to T (see FIG. 9A-B).







TABLE 2







A summary of the human cytidine


deaminase family of enzymes.










APOBEC-
HotSpot




3A
motif
Substrate
Function





AID
WRC
ssDNA
Somatic hypermutation





Class switch





recombination


 


Apo1
G/CTC
ssDNA
Lipid metabolism


 


A3A
T/CCA
ssDNA
Inhibits parvovirus,





HPV, retroelements


 


A3B
C/GTC
ssDNA
Inhibits HIV, HBV,





retroelements


 


A3C
TC/TC
ssDNA
Inhibits HBV, HPV,





retroelements


 


A3DE
WWC
ssDNA
Inhibits HIV


 


A3F
TTC
ssDNA
Inhibits HIV, HBV,





retroelement


 


A3G
CCC
ssDNA
Inhibits HIV, HBV,





retroelement


 


A3H
unknown
unknown
Inhibits HPV


 


Apo4
unknown
unknown
unknown









All references cited herein including U.S. application Ser. No. 13/827,885 filed Mar. 14, 2013 and U.S. Provisional Application No. 61/611,295 filed Mar. 15, 2012 are incorporated by reference.
EXAMPLES
Example 1: Synthesis of APOBEC-3A, AID and Mutants Thereof Using PURExpress
The AID and APOBEC-3A DNA sequences were codon optimized and reverse synthesized to form the gene sequence. The synthesized DNA was subcloned by TOPO® TA Cloning® (Life Technologies, Carlsbad, Calif.) under a T7 phage promoter and inserted into NEB 5-alpha F′/q Competent E. coli (New England Biolabs, Ipswich, Mass.). Mutant proteins were produced with the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Mass.) before being subcloned as above. 200 ng of each plasmid was used as template for in vitro synthesis of AID, APOBEC-3A, or their mutants using PURExpress. The in vitro reaction was incubated at 37° C. for 5 hours, and the synthesized proteins were boiled for 3 minutes, and after precipitation by centrifugation, a sample of the supernatant was loaded on an SDS gel. The results are shown in FIG. 1.
Example 2: Biochemical Assay for Cytosine Deamination (See FIG. 2)







TABLE 3







The following components were combined in a 1X solution to convert


C to U:











Volume,




Reaction Component
μl
Stock
Final Concentration













Nuclease-free water
15.8

Total volume: 20 μl












Oligonucleotide
1
1
μM
50
nM


RNase A
0.2
10
mg/ml
1
μg










NEBuffer 1 (New
2
10x
1x


England Biolabs,


Ipswich, MA)











PURE extract
1

50
ng


(APOBEC-3A/AID









The oligonucleotide was synthesized with a FAM label (Integrated DNA Technologies, Coralville, Iowa). The reaction mixture was incubated at 37° C. for 1 hour, and 10 μl of 1× NEBuffer 1 were added, containing 0.5 μl (0.5 U) of USER, and incubated additional 30 minutes at 37° C.
The products were separated on a 15% TBE-Urea gel (Invitrogen, Grand Island, N.Y.) using the XCell SureLock® Mini-Cell (Invitrogen, Grand Island, N.Y.).
Serial dilutions were performed on samples in order to determine the activity of AID, APOBEC-3A, or their mutants in a fixed ratio (such as 1:1, 1:2, 1:4, 1:8, etc.) as indicated below:







TABLE 4







Components were combined in a 1X solution or a titration assay of the


selected deaminase










Reaction Component





w/o deaminase
Volume, μl
Stock
Final Concentration













Nuclease-free water
16.8

Total volume: 20 μl












Oligonucleotide
1
1
μM
50
nM


RNase A
0.2
10
mg/ml
1
μg










NEBuffer 1
2
10x
1x









1 μl of APOBEC-3A or AID (wild type or mutant) after in vitro transcription/translation using PURExpress (containing about 50 ng of DNA deaminase enzyme) was added to the first tube in the serial dilution (1:1) which then contained 40 μl of the reaction mixture with deaminase (1× solution). 20 μl from the first tube was placed into 20 μl of reaction components without enzyme in a second tube (1:2) and so forth for the desired numbers of dilutions resulting in 2×, 4×, 8×, 16× and 32× dilutions (1×) in a reaction volume of 20 μl. The reaction mixture was incubated at 37° C. for 1 hour, and 10 μl of 1× NEBuffer 1 were added, containing 0.5 μl (0.5 U) of USER, and incubate additional 30 min at 37° C. The products were separated on a 15% TBE-Urea gel using the XCell SureLock Mini-Cell.
Generation of Mutants
The APOBEC3A or AID mutants were generated by random mutagenesis or site-specific mutagenesis.
For random mutagenesis the error-prone PCR method according to Cirino, et al., Methods in Molecular Biology, 231:3-10 (2003), was used; although the MgCl2 concentration was increased to 7 mM concentration in the reaction.
For site-specific mutagenesis, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Mass.) was used following the manufacturer's instructions. Target residues were selected according to sequence conservation, or predicted location in loop domains (Kohli, et al., Journal of Biological Chemistry, 284.34:22898-22904 (2009)) The mutant proteins were manufactured in cell-free PURExpress® system (see Example 1) and tested for activity as described above in Examples 2 and 3.
Example 3: APOBEC-3A Activity Assay on 5-mC Containing Substrate
A. Biochemical Assay for 5-mC Deamination
The following components were combined in a 1× solution. The following components were combined in a 1× solution in order to convert 5-mC to T (and C to U) in the oligonucleotide.







TABLE 5







Reaction components for 5-mC deamination










Reaction Component
1x













Nuclease-free water
16.5



Oligonucleotide * (1 μM)
1



RNase A (100 μg/ml)
0.2



NEBuffer 1 (10x)
2



PURE extract
0.3





* TATGGGGAAGGTTAGGGAAGATAAGAATAGAATGAAT/iMe-dC/GAAGGATGAATATGAGGTGAGGAGTAGGATGGG (SEQ ID NO: 17)


iMe-dC = 5-methylcytosine






The reaction mixture was incubated at 37° C. for 12-16 hours, or overnight, and PCR reaction was performed followed by Msel digestion.







TABLE 6







PCR reaction components










Component
50 μl 1x















5X Epimark ® HS Taq Reaction Buffer (New
10
μl



England Biolabs, Ipswich, MA)



10 mM dNTPs
1
μl



10 μM Forward Primer
1
μl



10 μM Reverse Primer
1
μl



Deaminated Oligonucleotide (1 μM)
1
μl



Epimark HS Taq DNA Polymerase
0.25
μl



Nuclease-free water
35.75

















TABLE 7







PCR cycling protocol











Cycling Step
Temp
Time
















Initial denaturation
95° C.
1
minute



Denaturation
95° C.
30
seconds



Annealing
56° C.
30
seconds



Extension
68° C.
20
seconds



Final extension
68° C.
5
minutes











Number of cycles
25 cycles

















TABLE 8







Msel digestion










Reaction Component
20 μl 1x














PCR product
8



Nuclease-free water
9.7



NEBuffer 4
2



Msel 10 u/μl
0.3










The reaction mixture was incubated at 37° C. for 0.5 hours and the DNA digestion products were analyzed on 2% agarose gel (see FIG. 7A-D). The 5-mC was converted to T and the PCR product formed after this conversion could be cleaved by the restriction endonuclease Msel.
B. APOBEC-3A Activity Assay on U5mC Containing Substrate
1 μl of human APOBEC-3A from PURExpress system extract was serially diluted 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and reacted with 1 pM ssDNA (TATGGGGAAGGTTAGGGAAGATAAGAATAGAATGATUmCTAAGGATGAATATGAGGTGAGGAGTAGGATGGG (SEQ ID NO:17) in NEBuffer 1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 1 mM Dithiothreitol pH 7.0) in the presence of RNaseA (1 μg). Reactions were incubated for 14 hours (overnight) at 37° C. Deaminations were detected through PCR reaction (FW primer: 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGTATGGGGAAGGTTAGGGAAG (SEQ ID NO:18), REV primer: 5′-CCTCTCTATGGGCAGTCGGTGATCCCATCCTACTCCTCACCTC (SEQ ID NO:19)) and digestion with Msel restriction endonuclease as described in Example 3 and shown in FIGS. 7C and 7D.
Example 4: APOBEC-3A Activity Assay on 5-hmC Containing Substrate
1 μl of APOBEC-3A from PURExpress extract was reacted with 1 pM fluorescein (F)-labeled ssDNA in NEBuffer 1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 1 mM Dithiothreitol pH 7.0) in the presence of RNaseA (1 μg). Reactions were incubated for 60 minutes at 37° C. Deaminations were detected through breakage of DNA at abasic sites generated by 0.5 μl of each of SMUG1 (5 u/μl) and EndoVIII (10 u/μl) enzyme. The results are shown on a urea gel and show no conversion of 5-hmC to U except using when using the control oligonucleotide containing 5-hmU and SMUG1/EndoVIII which produces the second band. The results are shown in FIG. 8A-B in which none of the 5-hmC is observed to be converted to 5-hmU.
Example 5: APOBEC-3A Deaminates 5m-C but not 5-hmC in Bisulfite Converted DNA
A DNA substrate of 489 bp long containing four CpG sequences at positions 80, 174, 239 and 356 was methyl in vitro with SssI methylase (New England Biolabs, Ipswich, Mass.). Methyl substrate was bisulfite converted using EpiMark Bisulfite Conversion Kit, amplified using FW GAGGAGGAAAAGAGAAATGGAGTGTGG (SEQ ID NO:20) and REV CTCACACCTCTCCTCTCACTCAC (SEQ ID NO:21) primers and EpiMark HS Taq DNA Polymerase. PCR fragment was inserted into TOPO TA Cloning vector and transformed into NEB 5-alpha F′/q Competent E. coli. When tested by sequencing, it was found that 100% of 5-mCs were identified as C.
An aliquot of the bisulfite converted DNA sample was treated with cytidine deaminase, or a mixture of cytidine deaminases for 4 hours to convert all 5-mCs to Ts (described in Example 1). After the reaction was completed, the deaminated DNA was amplified using FW GAGGAGGAAAAGAGAAATGGAGTGTGG (SEQ ID NO:20) and REV CTCACACCTCTCCTCTCACTCAC (SEQ ID NO:21) primers and Epimark HS Taq DNA Polymerase. PCR fragment was inserted into TOPO TA cloning vector and transformed into NEB 5-alpha F′/q Competent E. coli. When tested by sequencing, it was found that 100% of 5-mCs were identified as Ts. The results are shown in FIGS. 11 and 12.
Example 6: Discrimination Between 5-mC and 5-hmC Using Cytidine Deaminase and DNA Oxygenase Enzymes
Bisulfite sequencing in Example 5 was replaced here by sequential oxygenase and deaminase treatment.
A. Use of TET as the Oxygenase
The oxygenase reaction was performed as follows: 100 ng of NIH 3T3 Mouse Genomic DNA (New England Biolabs, Ipswich, Mass.) was incubated with TET1 (WiseGene, Chicago, Ill.) in 50 μl of reaction mixture for 3 hours. DNA was cleaned up using Zymo Clean & ConcentratorTM kit (Zymo Research, Irvine, Calif.).
TET is active on dsDNA. 50 μl of TET treated and untreated NIH 3T3 Mouse Genomic DNA in two separate tubes were heated up to 99° C. for 5 minutes, spun down briefly and put on ice.
B. Use of mYOX1 Instead of TET as the Oxygenase
In this example, a double-stranded substrate, containing 5-mC was oxidized with mYOX1. mYOX1 is active on ssDNA.
The sequence of the substrate was:









(SEQ ID NO: 22)



CGGCGTTTCCGGGTTCCATAGGCTCCGCCCXGGACTCTGATGACCA



GGGCATCACA



(X = 5 - mC)













TABLE 9







Reaction components for reaction of oligonucleotide with mYOX1










Reaction Component
Volume, μl
Stock
Final Concentration














ddH2O
3

to 20
μL


Bis-Tris pH 6.0
1
1M
50
mM


NaCl
1
1M
5
0 mM












DTT
1
20
mM
1
mM


Ascorbic acid
2
20
mM
2
mM


α-ketoglutarate
2
10
mM
1
mM


FeSO4
1
2
mM
100
μM


Oligonucleotide
4
10
μM
2
μM


MYOX1
5
16
μM
4
μM









The sequence for mYOX1 is as follows:










(SEQ ID NO: 23)



MTTFKQQTIKEKETKRKYCIKGTTANLTQTHPNGPVCVNRGEEVAN



 



TTTLLDSGGGINKKSLLQNLLSKCKTTFQQSFTNANITLKDEKWLK



 



NVRTAYFVCDHDGSVELAYLPNVLPKELVEEFTEKFESIQTGRKKD



 



TGYSGILDNSMPFNYVTADLSQELGQYLSEIVNPQINYYISKLLTC



 



VSSRTINYLVSLNDSYYALNNCLYPSTAFNSLKPSNDGHRIRKPHK



 



DNLDITPSSLFYFGNFQNTEGYLELTDKNCKVFVQPGDVLFFKGNE



 



YKHVVANITSGWRIGLVYFAHKGSKTKPYYEDTQKNSLKIHKETK






The reaction mixture was incubated at 34° C. for 1 hour.
1 μL of Proteinase K (20 mg/mL−1) (New England Biolabs, Ipswich, Mass.) was added to the reaction mixture and incubated for 1 hour at 50° C. DNA was isolated using the QIAquick® Nucleotide Removal Kit (Qiagen, Germany) and samples were analyzed by LC/MS.
These conditions cause 5-mC to be oxidized to 5-hmC.
Oxidation with mYOX1 of Mouse NIH 3T3 Genomic DNA







TABLE 10







Reaction components for oxygenase treatment of genomic DNA










Reaction Component
Volume, μl
Stock
Final Concentration














ddH2O
15

to 50
μl


Bis-Tris pH 6.0
2.5
1M
50
mM


NaCl
2.5
1M
50
mM












DTT
2.5
20
mM
1
mM


Ascorbic acid
5
20
mM
2
mM


α-ketoglutarate
5
10
mM
1
mM


FeSO4
2.5
2
mM
100
μM


NIH 3T3 DNA
10
200
ng/μL
2
μg


mYOX1
5
200
μM
20
μM









Human APOBEC-3A (100 ng from example 1), RNaseA (1 μg) were combined with ssDNA substrate (oxidated and non-oxidated NIH 3T3 Mouse Genomic DNA in separate tubes) and incubated in the reaction buffer (NEBuffer 1, (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 1 mM Dithiothreitol pH 7.0)) at 37° C. for 12-16 hours. Reactions were terminated by incubating at 80° C. for 10 minutes.
The oxidized and deaminated DNA or deaminated DNA only were PCR amplified and cloned. 2 μl from each sample (TET1+Deaminase and Deaminase only) were used for the PCR reaction using Epimark HS Taq DNA Polymerase. Primers were designed for deaminated DNA (all Cs become Ts, except oxidized 5m-C or 5hm-C). PCR products were visualized on 1.5% agarose gel and cloned into the TOPO TA Cloning vector and transformed into NEB 5-alpha F′/q Competent E. coli. DNA was isolated from individual colonies and sequenced using nested primers.
The sequence results were interpreted as follows:
(a) Original sequence: GGGTmCGGACCGhmC (SEQ ID NO:7)
(b) After TET and deaminase treatment:GGGTCGGATTGC (SEQ ID NO:11)
(c) After deaminase only treatment: GGGTTGGATTGC (SEQ ID NO:12)
After alignment of sequences, TET and deaminase treatment (b) and deaminase only treatment (c) show Cs that corresponds to 5-hmC but not C. After TET and deaminase treatment only, C also corresponds to 5-mC but after deaminase treatment only, 5-mC corresponds to T.

## Claims

1. A composition comprising a protein variant having at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprising an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1;

(b) SEQ ID NO:2 and comprising an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not a Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2, or

(c) SEQ ID NO:3 (activation induced cytidine deaminase (AID)) and comprising a deletion at a position corresponding to position 117 of SEQ ID NO: 3.

2. A composition according to claim 1, further comprising at least one of a purified oxygenase, a polymerase, a polynucleotide and/or at least one primer and dNTPs.

3. An in vitro mixture comprising a protein variant of claim 1 and a purified oxygenase.

4. An in vitro mixture according to claim 3, wherein the oxygenase is a methyl pyrimidine oxygenase or a 5-methylcytosine oxygenase.

5. An in vitro mixture according to claim 3, wherein the protein variant has at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprises an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. a Trp at the position corresponding to position 45 in SEQ ID NO:1,
ii. a Gln at the position corresponding to position 109 in SEQ ID NO:1, and
iii. aft His at the position corresponding to position 123 in SEQ ID NO:1, or

(b) SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an Asn at the position corresponding to position 23 in SEQ ID NO:2,
ii. a Val at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an Arg at the position corresponding to position 69 in SEQ ID NO:2.

6. An in vitro mixture according to claim 4, wherein the methyl pyrimidine oxygenase is mYOX.

7. An in vitro mixture according to claim 4, wherein the 5-methylcytosine oxygenase is TETI.

8. An in vitro mixture, comprising a purified cytidine deaminase and a purified DNA polymerase, wherein the purified cytidine deaminase has at least 90% sequence identity to:
(a) SEQ ID NO:1 and comprises an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1,

(b) SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2, or

(c) SEQ ID NO:3 and comprises a deletion at a position corresponding to position 117 of SEQ ID NO: 3.

9. An in vitro mixture according to claim 8, further comprising a polynucleotide and at least one primer and dNTPs.

10. A method for determining for a cytidine deaminase, a cytosine preference according to an adjacent nucleotide, comprising:
(a) reacting a polynucleotide containing a cytosine with a protein variant of claim 1 to convert the cytosine to uracil where the cytosine may be adjacent to any of adenine, guanine, thymine, uracil or cytosine;
(b) reacting the product of (a) with a glycosylase and an AP endonuclease so as to cleave the polynucleotide at the uracil;
(c) detecting the cleavage product from (b) to determine the activity of the cytidine deaminase for the cytosine adjacent to any of adenine, guanine, thymine, uracil or cytosine.

11. A method according to claim 10, wherein the polynucleotide is single stranded.

12. A method according to claim 11, wherein the polynucleotide is labeled at one end.

13. The composition of claim 1, wherein the protein has at least 90% sequence identity to SEQ ID NO:1, an Asp at the position corresponding to position 180 in SEQ ID NO:1 and:
i. an amino acid that is not Ser at the position corresponding to position 45 in SEQ ID NO:1,
ii. an amino acid that is not Glu at the position corresponding to position 109 in SEQ ID NO:1, and
iii. an amino acid that is not Arg at the position corresponding to position 123 in SEQ ID NO:1.

14. The composition of claim 1, wherein the protein has at least 90% sequence identity to SEQ ID NO:2 and comprises an Arg at the position corresponding to position 29 in SEQ ID NO:2, an Arg at the position corresponding to position 104 in SEQ ID NO:2, an Arg at the position corresponding to position 123 in SEQ ID NO:2 and:
i. an amino acid that is not Thr at the position corresponding to position 23 in SEQ ID NO:2,
ii. an amino acid that is not Gly at the position corresponding to position 25 in SEQ ID NO:2, and
iii. an amino acid that is not Cys at the position corresponding to position 69 in SEQ ID NO:2.

15. The composition of claim 1, wherein the protein has at least 90% sequence identity to SEQ ID NO:3 and comprises a deletion at a position corresponding to position 117 of SEQ ID NO: 3.