# US 10,370,706 B1 · Molecular recognition systems with pyrimidine analog pairing

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- Number: US 10,370,706 B1
- Document ID: US10370706B1
- Status: Granted
- Kind: B1
- Application number: 16/111,590
- Filed: 2018-08-24
- Granted: 2019-08-06
- Earliest claimed: 2017-03-16
- Applicants: Shuichi Hoshika; Nicole A Leal; Steven A Benner
- Inventors: Shuichi Hoshika; Nicole A Leal; Steven A Benner
- Web page: https://patentgrep.com/patent/US10370706B1

## CPC

- C12Q 1/6853: using modified primers or templates
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6813: Hybridisation assays
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2533/101: Primer extension
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

This invention covers a new molecular recognition system, where duplexes of DNA-like molecules comprise segments built from nucleotides that carry only a small pyrimidine-like analog, and where the segments pair by pyrimidine analog:pyrimidine analog “skinny” pairing. This pairing retains hydrogen bonding complementarity. Further, this invention relates to processes for preparing those duplexes, and processes that use such duplexes as primer:template complexes for reactions catalyzed by DNA polymerases.

## Claims

### Claim 1

A composition, wherein said composition comprises a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide comprises a segment composed entirely of nucleotides carrying heterocycles selected independently from the group consisting of
and wherein said second oligonucleotide comprises a segment composed entirely of nucleotides comprising heterocycles selected from the group consisting of
wherein Su indicates the point of attachment of said heterocycle to a sugar of said first or second oligonucleotide, R′ is CH3, alkyl, alkenyl, alkynyl, or alkyl, alkenyl, or alkynyl carrying a functional group, wherein R is H, CH3, alkyl, alkenyl, or alkynyl, or functionalized alkyl, alkenyl, or alkynyl, and X is N or C—NO2, wherein said segments form a duplex region in which said segments are joined by hydrogen-bonding between complementary heterocycles forming skinny pairs, wherein
and wherein said duplex region comprises at least three such pairs.

### Claim 2 (depends on 1)

The composition of claim 1, wherein said functional group is an amino group or a thiol group.

### Claim 3 (depends on 1)

The composition of claim 1, wherein said heterocycles are independently selected from the group consisting of

### Claim 4

A process for forming a molecular complex, said process comprising contacting a first oligonucleotide with a second oligonucleotide in aqueous solution, wherein said first oligonucleotide comprises a segment comprising entirely nucleotides carrying heterocycles selected independently from the group consisting of
wherein said second oligonucleotide comprises a segment comprising entirely nucleotides carrying heterocycles selected independently from the group consisting of
wherein Su indicates the point of attachment of said heterocycle to a sugar of said first or second oligonucleotide, R′ is CH3, alkyl, alkenyl, alkynyl, or alkyl, alkenyl, or alkynyl carrying a functional group, wherein R is H, CH3, alkyl, alkenyl, or alkynyl, or alkyl, alkenyl, or alkynyl carrying a functional group, and X is N or C—NO2, wherein said segments form one or more duplex regions, wherein said segments within said duplex region(s) is (are) joined by hydrogen-bonding between complementary heterocycles forming skinny pairs, wherein
wherein said duplex region comprises at least three such pairs.

### Claim 5 (depends on 4)

The process of claim 4, wherein said functional group is an amino group or a thiol group.

### Claim 6 (depends on 4)

The process of claim 4, wherein said heterocycles are independently selected from the group consisting of

### Claim 7 (depends on 4)

The process of claim 4, whrein said aqueous solution is between 20° C. and 40° C.

### Claim 8 (depends on 4)

Th process of claim 4, wherein said aqueous solution has a pH between 5 and 9.

### Claim 9

A process for extending a primer using a polymerase, said process comprising contacting a polymerase with a duplex comprising a template oligonucleotide and a primer oligonucleotide,
wherein the template oligonucleotide comprises at least two segments, including a first segment at or near its 3′-end consisting of nucleotides carrying heterocycles selected independently from the group consisting of
and a second segment positioned 5′-distal to the first segment and comprising any nucleotides,
and wherein the primer oligonucleotide is complementary to part or all of the 3′ first segment of the template oligonucleotide, and the 3′-end of the primer oligonucleotide comprises a segment consisting of nucleotides carrying heterocycles selected independently from the group consisting of
wherein Su indicates the point of attachment of said heterocycle to a sugar of said template oligonucleotide or said primer oligonucleotide, wherein R′ is CH3, alkyl, alkenyl, alkynyl, or alkyl, alkenyl or alkynyl carrying a functional group, wherein R is H, CH3, alkyl, alkenyl, alkynyl, or functionalized alkyl, alkenyl or alkynyl, and X is N or C—NO2,
wherein the 3′ template segment and the 3′ primer segment form one or more duplex regions, wherein segments within said duplex region(s) are joined by hydrogen-bonding between complementary heterocycles to form skinny pairs, wherein each said skinny pair is independently selected from the group consisting of
wherein at least one said duplex region comprises at least three skinny pairs.

### Claim 10 (depends on 9)

The process of claim 9 wherein said polymerase is a Klenow fragment of DNA polymerase I or a Stoffel fragment of a DNA polymerase from Thermus aquaticus.