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Joa's White
No. 226
Joa's White was named after our original Colour Consultant who developed many of the Farrow & Ball colours. It is a very warm neutral with the merest hint of underlying black which makes it perfect to combine with the limestone, leather and linens so often used in classic contemporary homes. Joa's White is a warmer and more contemporary version of the classic Off-White. Mid Tones Undercoat.
Joa's White 21 21
Beautiful overall effect The colour is stunning and adds depth to the room. It is also lovely to apply. 03 June 2015
Good quality and longevity An excellent quality paint with good wearing record and can easily be cleaned. First class colour choice. 15 May 2015
light and airy I love this color, my husband hates white paint but this is a beautiful tint of cream with a hint of yellow, it really lightened up the living room. I am going to carry this color into the kitchen 03 May 2015
Great paint. Well worth the price. I used this to pant directly onto the less than perfect bare plaster walls in a bedroom in our Georgian house. 3 coats later it looks great and has a surprisingly uniform finish given what's underneath. We used £150 of F&B paint but the end result was so much better than we've achieved in other rooms with cheaper brands that I'd say it was well worth it. 04 February 2015
Nice upgrade from magnolia We had typical magnolia everywhere and wanted a not too dramatic change , Joa's White worked perfectly. We went for a contrast with white on the rear wall and chimney breast , looks great and the quality of the paint is obvious to see. Highly recommend this colour . 29 December 2014
Matched perfectly in my kitchen Rich latte colour looks perfect against oak kitchen top and off white cupboards 27 September 2014
Looks great in West facing room but slightly too peach in our East facing room We wanted a white that was not yellow or green or cold or bright- something with a warmth to it. Joa's looks lovely in the east facing bedroom but is too peach for my liking in the West facing room. It almost looks pink. I can imagine it looking great in a Cotswold stone house or even by the seaside in a room flooded with light but in our Victorian town house it doesn't quite work. I wanted sandy as opposed to peachy. We are using skimming stone in the rest of the house so it will be interesting to compare. I have a feeling we may replace the Joa's with skimming stone in this room. Otherwise, used Dimity on the ceiling and pointing on the woodwork, both of which are absolutely gorgeous. Dimity is greyer than I thought it would be but just lovely- even our decorator said what a beautiful ceiling colour it was and how refreshing it was that we had not gone for plain white. 31 August 2014
Superb product As usual this Farrow & Ball paint is a pleasure to use and I wouldn't consider using any other brand. 29 August 2014


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WEBSITE OF THE DAY - babyfriendlyboltholes.co.uk
Ah, the good old British self-catering holiday. Dodgy plumbing, grimy kitchen utensils and a faint musty smell that permeates everything you unpack and never quite goes away again.
Not so with this site.
Each property listed here has been rigorously assessed for quality, style and, above all, family friendliness.
These are not your run-of-the-mill properties, so don't expect run-of-the-mill prices. But if you're looking for somewhere to spoil yourselves and the kids this summer, this is a great place to look.
There are UK properties and also some in France, Italy, Greece and Spain, with accommodation available for parties of two to more than 30.
It's about time somebody paid this overlooked corner of the holiday market proper attention, and it looks like babyfriendlyboltholes are the pioneers.
If you have a website that you want to tell us about email us via the feedback form.

subset: commoncrawl


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We have rich conversations with our clients every day, and increasingly these conversations are happening online. We encourage you to ask questions, share your views, and connect with like-minded professionals.
The International Annual Review demonstrates our achievements and reflects the insights and expertise professionals bring to client relationships.
KPMG's Global IFRS Institute provides information and resources to help Board and Audit Committee Members, Executives, Management, Stakeholders and Government Representatives gain insight on the evolving global financial reporting framework.
KPMG's global network of climate change and sustainability practices helps you build long-term value in a rapidly changing world.
Mining organizations need to adapt across the mining asset lifecycle, from expansion to closure, as changing economic, political and regulatory environment demand greater flexibility.
The Chemical industry's landscape is changing fast - global companies are developing or launching new products and services – and appear optimistic - setting sights on geographic expansion.
Over the last two decades many industries have changed their value proposition by developing their customers' capacity to create value.
KPMG Global Manufacturing Outlook 2014 surveyed 460 executives worldwide, and offers practical and provocative insights to help organizations evaluate future strategies.
The global job search tool allows you to search available career opportunities within many of our 150 member firms worldwide.
KPMG's International Case Competition (KICC) is a chance for students to challenge yourself to solve a real business issue and an opportunity to meet new people across the globe.
Traditional risk management methodologies also underestimate systemic risk caused by an increasingly interconnected world. These interdependencies have a contagious effect across countries and financial systems, making crises deeper and recovery slower.
Drawing on personal experience
It is no longer enough to simply crunch numbers; risk models should also incorporate the insights of senior managers that have experienced crises first hand. And risks cannot be looked at in isolation. They tend to combine with each other in "risk clusters" that heighten their impact. Regulators are increasingly seeking more sophisticated risk analyses in stress and scenario testing, particularly for going concern analyses and capital adequacy assessments.
Predicting the next crisis
Today's financial systems are extremely complex, due to globalization, market liberalization and technology. This also makes them more unpredictable, so organizations need systemic risk models and associated controls that:
Does your organization:
routinely report systemic risk to risk committees and the board?
employ systemic risk models that embrace current data and future predictions?
produce risk cluster analyses?
To discuss these questions further, please contact:
Andries Terblanche
KPMG in Australia
+61 2 9335 7570
Jacinta Munro
KPMG in Australia
+61 3 9288 5877
To read the full article, download the PDF.
© 2015
KPMG International Cooperative ("KPMG International"), a Swiss entity. Member firms of the KPMG network of independent firms are affiliated with KPMG International. KPMG International provides no client services. No member firm has any authority to obligate or bind KPMG International or any other member firm vis-à-vis third parties, nor does KPMG International have any such authority to obligate or bind any member firm. All rights reserved.


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1. In a pan, heat olive oil on medium. Add the onions and sauté till translucent.
2. Put the turkey and cook till the meat turns brown. Drain off excess fat rendered.
3. Add liquid smoke and mix well. Cook for a few more minutes.
4. Put the tomato sauce, barbecue sauce, ketchup, Worcestershire sauce and stir well till incorporated.
5. Season with celery flakes and garlic pepper seasoning. Stir to mix and continue to cook till the sauce thickens to desired consistency. Turn off the heat.
6. In a serving plate, serve in toasted potato buns with baked fries, salad or corn on the cob.


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Vice President Joe Biden speaks during a campaign stop at the Bridge View Center, Tuesday, Sept. 18, 2012, in Ottumwa, Iowa. (AP Photo/Charlie Neibergall)
WASHINGTON: US Vice President Joe Biden pressed Iraq's Prime Minister Nuri al-Maliki in a telephone call Friday to bar the passage of weapons shipments to Syria through Iraqi airspace, the White House said.
"The vice president and the prime minister addressed issues of regional security, including the need to prevent any state from taking advantage of Iraq's territory or air space to send weapons to Syria," the White House said.
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Audio Samples
Album Summary
- Mstislav Rostropovich (Cello)
- Rudolf Barshai (Viola)
- Leonid Kogan (Violin)
Notes & Reviews:
"And there's freedom aplenty in these vigorous, highly charged performances...These are strong-jawed readings with a great sense of purpose...Another asset to this set is the violinist himself, Leonid Kogan, whose tone is searing in its intensity...this new release captures a compelling moment in history: it must have been some evening!" -Gramophone
This CD, the latest title within the acclaimed Russian Masters series, presents three towering Russian musicians. At a superb 1960s Prague Spring festival concert, Kogan, Barshai and Rostropovich performed together three of Beethoven's four string trios. These three soloists encounter on a single stage was a veritable feast for chamber music lovers and one of the apices of the Prague Spring. Now, half a century later, you too can savor this remarkable concert.
Three legends on one stage - a precious encounter you can again enjoy, half a century on.
Recording information: 15th Spring International Music Festival at the Rudolfi (06/02/1960).
ReviewsThere are currently no reviews, be the first one!
Also Purchased
Works DetailsBeethoven, Ludwig van : Trio for Violin, Viola and Cello no 3 in G major, Op. 9 no 1
- Performers: Mstislav Rostropovich (Cello); Rudolf Barshai (Viola); Leonid Kogan (Violin)
- Running Time: 24 min. 11 sec.
- Period Time: Classical
- Written: 1797-1798
- Studio/Live: Live
Beethoven, Ludwig van : Trio for violin, viola & cello in C minor, Op. 9/3
- Performers: Leonid Kogan (Violin); Mstislav Rostropovich (Cello); Rudolf Barshai (Viola)
- Notes: Composition written: 1797-98.
- Running Time: 21 min. 14 sec.
- Period Time: Classical
- Form: Chamber Music
- Written: 1797-1798
- Studio/Live: Live
Beethoven, Ludwig van : Trio for violin, viola & cello in E flat major, Op. 3
- Performers: Mstislav Rostropovich (Cello); Leonid Kogan (Violin); Rudolf Barshai (Viola)
- Running Time: 35 min. 11 sec.
- Period Time: Classical
- Written: circa 1794
- Studio/Live: Live


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Integral Master Coach™
Life is complex. Time is tight. Circumstances limiting. The gap between your vision and reality thus grows. Meaningful change happens faster with a coach, so lets close that gap & make some cool things happen!
Ryan Leech Connection
Your online yoga & coaching studio for mountain bikers with weekly on & off bike practices to optimize your riding performance & experience.
Professional Mountain Biker
Performing Shows at Special Events and Schools, and promoting a healthy mountain biking lifestyle with the support of my sponsors. How can I be of service, I love new projects and ideas that leverage my riding skills and reputation for positive vibes and change!
The Power of Belief in Practice
Wheelies took a very long time for me to learn. It was one of the very first tricks I worked on with my first mountain bike. I practiced relentlessly to learn it. Once I overcame my frustrations and finally learned to wheelie, I had the belief that I could learn other technical bike tricks. I […]
Mindfulness in Adventure
Soon to be published in Explore Magazine: 4 Steps to bring mindfulness to your next adventurous pursuit: Bring awareness to your breath Seemingly simple, and seriously profound. Yet reliable access to the focus that breath awareness brings is not simple, it's a skill, and requires an ability to become more quickly aware of those times […]
The word 'retreat' reminds me of being a kid during friendly (and sometimes not so friendly!) battles, where a strategy goes wrong and we'd run away yelling "RETREAT RETREAT!!" Same is true today, though my ego is far more stubborn and doesn't usually retreat soon enough! Retreating is the healthiest path in many circumstances, that's why the […]


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For some reason, women in comics almost always seem to have the most uncomfortable costumes. These superheroines are often forced to wear...More →
In our dreams, these are the cereals we would be able to eat for breakfast each morning. Artists Crystal Fontan a.k.a Bamboota...More →
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Drake Tsui is better known in the Deviant Art community under the moniker of Quirkilicious, and his delightful brand of rainbow colored...More →
German artist Micheal Kutsche is an award winning master of the visual arts. His astoundingly lifelike work breathes life into alternate realities...More →
Boss who-gic? So glad you asked! BossLogic (or the LGX as he is known on Deviant Art) is the code name of...More →


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Daily Sales Up to 70% OFF Limited-time deals start every day at 12 pm ET.
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Room Magic Garden Changing Pad Cover
Room Magic Garden Changing Pad Cover
Weights & Dimensions
|Product Type: Changing Pad Cover
|Material: Cotton
|Country of Manufacture: United States
About the ManufacturerRoom Magic, a new company in children's furniture and accessories, presents seven children's bedroom collections inspired by Matisse and a child's lively imagination: Bright, whimsical "cut outs" of tea cups, kissing fish and playful lizards animate brightly colored and natural finished birch wood designs. The designs have been so well received that Room Magic received a Pinnacle Award by the AAFD American Association of Furniture Designers.
Room Magic Garden Changing Pad Cover
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Tell us what you think about this product. It will help us provide you with better products in the future and help your fellow shoppers make better purchases.Write the first review
OUR SKU # ROM1124Garden Changing Pad Cover is also sometimes listed under the following manufacturer numbers: RM06-MG


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
45 year old man | Sherman Oaks, CA, USA | Seeking women 32-48 in California, United States
Active within 24 hours
Enjoying Every Minute
Looking to date someone that will eventually turn into a long-term relationship. I work as an Recording Engineer / Editor for a TV network, a music composer, play double Bassist/cello. I'm finishing another solo CD release. I write and listen to different styles of music. The most common genres Jazz, Classical, Latin and more. Originally from Detroit MI and moved to L.A. years ago. I'm close with my family and my background is Italian/French. I like to relax and hang out at my house. I like to have friends over for an occasional party or BBQ, Dinner ,bocci ball, shoot pool etc. I'm 6'1 and I like eating and staying healthy. I spend time outdoors Mt. Biking, hiking, composing music, working out, photography, cooking. I'm into the arts, music, museums, stuff around the house, weekend getaways, going out to dinner, sushi being one of my favorites.
Camping , Coffee and conversation , Cooking , Dining out , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Performing arts , Playing cards , Travel/Sightseeing , Wine tasting
Baseball, Basketball, Billiards / Pool, Bowling, Cycling, Football, Golf, Hockey, Martial arts, Other types of exercise, Swimming, VolleyBall, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I like Birds, Cats, Dogs, Exotic Pets, Fish, Gerbils / Guinea Pigs / Etc., Horses, Other, Reptiles
Some other viewpoint
I like to go out for dinner, sushi is my fav, movies, concerts, weekend getaways, Santa Barbara, Palm Springs.
After work I always find something to do. I like to go Mt. Biking, Hiking, I play Bass/Cello, working out, museums, billards, coffee, photography, music (all sorts), BBQ, hang with friends, or relax in the backyard or with a dvd, etc...
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'1" (185cms)
|5'0" (152cms) to 6'0" (182cms)
|Body type:
|Athletic and toned
|Slender, About average, Athletic and toned
|Black, Blue, Brown, Grey, Green, Hazel
|Light brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Occasionally
|Social Drinker
|No preference
|Technical / Science / Computers / Engineering
|No preference
I work for a TV network editing/recording different shows. Also a music composer and musician.
|I'll tell you later
|No preference
|Never Married
|No preference
|Have kids:
|No preference
|Wants kids:
|I'll tell you later
|No preference
|I'll tell you later
|Asian, Black / African descent, East Indian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian, Other
|Christian / Other
|No preference
|Bachelors degree
|No preference


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Tactical Pants - Galls
A popular choice for public safety professionals, the Galls Tactical Pants are...
Although the Daniel Defense M4, or DDM4, is a factory rifle, it looks like it was built by a custom shop. This carbine ships with quad fore rails, a removable backup sight, Magpul MOE adjustable stock, Daniel Defense vertical fore grip, a flared/beveled trigger guard, a flared mag well, and a Magpul 30-round PMAG.
Tags: AR-Type Rifles, Daniel Defense


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March for life News
For the month of July, our March for Life Give Live challenge is to host a "baby shower" for your local Pregnancy Resource Center! Continue reading
Legislation Retains Critical Focus on Humanity of the Child Washington, D.C – The U.S. House of Representatives voted 240 – 186 Wednesday to pass a bill that would ban abortions after 20 weeks of pregnancy."Thank you to the many … Continue reading
The House of Representatives will vote on a bill this week that would ban nearly all late-term abortions. Continue reading
Our Give Life Challenge for the month of May is to encourage moms raising kids with special needs! Would you sign a heartening card for moms of children with Down Syndrome? Continue reading
March for Life President Jeanne Mancini, along with a group of national pro-life leaders, released a joint statement urging House Republican Leadership to schedule a vote on the Pain-Capable Unborn Child Protection Act. Continue reading
Our Give Life Challenge for April is to share information about post-abortion healing. There are many things you can do and resources you can share to give life and hope to those in need. Continue reading
The March for Life mourns the passing of two pro-life champions, who went home to God this past weekend, Dr. Jack Wilke, and Brother Paul O'Donnell. Continue reading


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Rio Rico Homes for Rent
BusinessWeek "Best of the Web" Winner 3 Years Running
Search Rio Rico Home Rental Market
Since our family moves around a lot, we are always looking for rental properties. MyNewPlace has the most reliable search on the internet. We always find what we need. - Rio Rico,AZ
Popular Rental Properties in Rio Rico, Arizona
317-A S Paseo Tierra
Green Valley, Arizona, 85614
2 Bedrooms
3030 S Camino Kino
Green Valley, Arizona, 85622
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Green Valley, Arizona, 85614
2 Bedrooms
Rio Rico Homes for Rent
Are you considering a Rio Rico home rental? You can find a lot of enticing places to rent there and the exact right one is waiting for you - just start searching on MyNewPlace. Do you find yourself wishing you had a garage? A home rental can solve this problem. Whether you prefer condos, townhomes, or single family homes you can find the exact right rental house in one of the best parts of Rio Rico.
Want the inside scoop on Rio Rico? Check out our Rio Rico Local Talk for local tips and information about Rio Rico. You can also get answers to your Rio Rico questions from our local experts by using the "Ask a Question" feature.
Houses for Rent in Cities Near Rio Rico, AZ
MyNewPlace can help you find the Rio Rico, AZ home rental that will work best for you. Using our rental home search, you can view townhomes or condos for rent if you want to live downtown, or check out the rental listings for single family house rentals in the surrounding areas. Try one of these cities below for more rental houses near Rio Rico, AZ.


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"I was furious because I figured out in Europe that my real father had agreed to allow me to be adopted," he said.
A furious and frank debate over what to do with them follows, and the team agrees to let them go.
When New Jersey authorities learned of the sting, they were furious.
late 14c., from Old French furieus (14c., Modern French furieux), from Latin furiosus "full of rage, mad," from furia "rage, passion, fury." Furioso, from the Italian form of the word, was used in English 17c.-18c. for "an enraged person," probably from Ariosto's "Orlando Furioso."


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David Meerman Scotts marketing bible has become a modern day business classic.
This is the book every ambitious, forward-thinking, progressive marketer or publicist has at the front of their shelf. Business communication has changed over the recent years. Creative ad copy is no longer enough. The New Rules of Marketing and PR has brought thousands of marketers up to speed on the changing requirements of promoting products or services in the new digital age. This is a one-of-a-kind, pioneering guide, offering a step-by-step action plan for harnessing the power of the Internet to communicate with buyers directly, raise online visibility, and increase sales. Its about getting the right message to the right people at the right time - for a fraction of the cost of a big-budget advertising campaign. This new, updated edition includes:
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European IP bulletin — December 2012
In this issue (inter alia): European Commission to review misleading and comparative advertising directive; Information Commissioner's office publishes report on enforcement of new cookie rules; Scrabble tile trade mark declared invalid.
Click on the link above to download this McDermott Will & Emery briefing.
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Shanghai's ground-breaking Pilot Free Trade Zone could mark the beginning of the long-awaited liberalisation of China's legal services sector.


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Plague [PG-16] OOC and SUs
View Single Post
February 14th, 2011 (01:27 PM).
Forever Shawol and Aileean
Join Date: Jan 2010
Location: Sinnoh
Gender: Female
Nature: Adamant
Posts: 2,082
The crazy is mostly with Nightmare, only because he is insane, ANYWAYS.
LOL, I just realized how much of a field day the paparazzi is going to have due to the fact that Miharu (The Kanto Champion) is trapped in a city FILLED WITH ZOMBIES AND A VIRUS THAT COULD TURN SOMEONE INTO A ZOMBIE! XDDD And Miharu could also use her incredible connections as well as her own power to get out. XD Not much the army can say to one of the most powerful (at the current time) trainers in the world.
Army: Oh, sorry, ma'am you can't leave.
Miharu (putting on her champion look): Everyone here may be in the army, but I have important business to take care of back in Kanto. Unless the army wants the Pokemon League Board on their arses, the army should let myself and my friends go.
Army: Uh...uh...
Miharu (raises an eyebrow): Exactly. (walks out of Balice City)
"I want to become the SHINee that will exist in your hearts and be remembered forever." - Leader Onew
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Originally written on Fox Sports North | Last updated 7/18/13
MINNEAPOLIS -- Ronny Turiaf's northern connections run deeper than meets the eye. Judging by the passion in his voice while speaking with reporters Thursday night, a whole heck of a lot deeper. "I'm so excited about it," the eight-year, bench-presence veteran said of signing with the Timberwolves, "I feel like I'm a rookie all over again." In reality, there's a lot of wear and tear on the 30-year-old's 6-10, 250-pound frame. Eight seasons of bouncing from city to city will do that. But president of basketball operations Flip Saunders brought Turiaf in for, if nothing else, his sheer fervor. Whether it's been spelling a starting center, handing out locker room advice or dancing on the sideline in celebration, that's never been in short supply. "The greatest thing about Turiaf is that he's got great enthusiasm, great love for the game," Saunders said recently. "He's got great passion." What's not as noticeable is that Turiaf actually played for Saunders during one of his journey's bri...
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Taking A Look At The Best Way To Brush Your Teeth
Story By: Larry Stine
Originally Published on: 8/17/2014
Brushing and flossing your teeth should be a part of your daily routine. That's because tooth decay can cause all kinds of problems, including abscesses and inflammation in the body that can lead to heart disease. But not every toothbrush is the same. What's the best way to tackle your teeth? Here's today's Health Minute. We all know that dental hygiene is extremely important, but sometimes our brushing habits could do more harm than good. "We recommend that you brush your teeth for two minutes, with a light to a medium amount of force," says Dr. Youssef Obeid, a prosthodontist. Whether it's electric or manual, it is very important to brush correctly and regularly. "What we recommend for a toothbrush is a soft or ultra soft, you know.," says Dr. Obeid. "We try to stay away from using a medium or a hard tooth brush nowadays because it will cause some damage for your own actual teeth." Electric does have its benefits, however. "People have a tendency to brush too hard, or too fast," says Dr. Obeid. "That's why sometimes the electric toothbrush will help you because it will give you the right amount of time, and will tell you if you are pushing too hard." In the end, the message is simple: "The best way to protect your teeth from cavities and from gum disease is still brushing your teeth," says Dr. Obeid.
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I hate that music she dances to. It sounds like a banjo that's badly in need of a tune.Originally Posted by Puffy
I hate that music she dances to. It sounds like a banjo that's badly in need of a tune.Originally Posted by Puffy
Makes all the routine posts.
Curious day-night OPS numbers for Ry-guy:
2003: day - .674
night - .831
2004: day - .849
night - .682
2005: day - .891
night - .674
There goes the hangover theory, I guess. But he can't seem to get away from a kind of Jekyll and Hyde thing. Geez, man, level out.
"Baseball is a very, very complex business. It's more of a people business than most businesses." - Bob Castellini
Dick Butkus had a quote- this is probably not exact but it went something like "in a third and short situation, I'll take the whiskey drinkers over the milk drinkers any day".
If Freel has a problemit is his problem, and if he can play, he can play.
I've been to dinner at Jimmy Buffet's house, and I've eaten it at a homeless shelter. And there's great joy and harrowing terror to be found in both places.
-Todd Snider
9191, if I didn't invite you along before we're all heading down to the bar to play some pool and suck some suds, you're welcome to come along.
Yep.Originally Posted by 919191
Generally, I don't trust the judgement of someone that won't sit down and have a drink occasionally anymore than I trust the judgement of someone that won't sit down and not have a drink occasionally.
/special cases & situations nonwithstanding
Kinda scary when you see that the REDS play only 33.95% of their 2006 games (55G) during the daytime.Originally Posted by lollipopcurve
Small market fan... always hoping, but never expecting.
Nah, the Reds dragged baseball into the reality of night baseball, it's one thing they should be proud of being a part of... that's an "image" not things like Freels arrest the other night.Originally Posted by RedFanAlways1966
Otherwise Cesar Cedeno never would have been a Red.
Once means that he only got caught once...twice means he's just plain stupid.Originally Posted by creek14
Rob Neyer: "Any writer who says he'd be a better manager than the worst manager is either 1) lying (i.e. 'using poetic license') or 2) patently delusional. Which isn't to say managers don't do stupid things that you or I wouldn't."
Board Moderators may, at their discretion and judgment, delete and/or edit any messages that violate any of the following guidelines: 1. Explicit references to alleged illegal or unlawful acts. 2. Graphic sexual descriptions. 3. Racial or ethnic slurs. 4. Use of edgy language (including masked profanity). 5. Direct personal attacks, flames, fights, trolling, baiting, name-calling, general nuisance, excessive player criticism or anything along those lines. 6. Posting spam. 7. Each person may have only one user account. It is fine to be critical here - that's what this board is for. But let's not beat a subject or a player to death, please.


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Dr. Yuen Yee specializes in gastroenterology, internal medicine in Bremerton, Washington.
Dr. Yuen Yee has been compared 263 times. Details of Dr. Yee's experience as an MD and his hospital affiliation are available on your free doctor report.
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Dr. Yuen Yee has been identified as specializing in Gastroenterology and Internal Medicine.
He obtained board certification from The American Board of Internal Medicine for Internal Medicine.
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Dr. Yuen Yee accepts the following plans at 2771 Hemlock St, Bremerton, WA
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Originally Posted by lewis-richards
one of the cameras can go up to 6X8 aswell?
The RB, but you need the accessories to do it, and you don't get to frame the shot entirely on the GG.
"Truth and love are my law and worship. Form and conscience are my manifestation and guide. Nature and peace are my shelter and companions. Order is my attitude. Beauty and perfection are my attack."
- Rob Tyner (1944 - 1991)


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Jul 4 @ 9:30 am – 12:00 pm
Last year we accomplished 1000 pullups over the course of the parade between 6 people. This year we will be using HUMAN horse power, built by CrossFit, to pull a Jeep Wrangler through the duration of the parade with harnesses attached to our shoulders.
Join us by walking in the parade with us and hand out frisbees and help us pull the Jeep!


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Additional information coming soon!... More
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text:
Methods are described for the production of nucleic acid ligands to the HIV-1 tat protein. Motifs I, II and III in FIG. 3 are nucleic acid ligands identified by the disclosed methods.
1. A nucleic acid ligand to the HIV-1 tat protein produced according to a method comprising:
a) preparing a candidate mixture of nucleic acids;
b) contacting the candidate mixture with the HIV-1 tat protein, wherein nucleic acids having an increased affinity to the HIV-1 tat protein relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and
d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids an increased affinity to the protein; whereby nucleic acid ligands of the HIV-1 tat protein are produced.
2. The nucleic acid ligand of claim 1 being a single stranded nucleic acid.
3. The nucleic acid ligand of claim 1 being a single stranded RNA.
4. A purified and isolated non-naturally occurring nucleic acid ligand to the HIV-1 tat protein.
5. The nucleic acid ligand of claim 4 wherein said ligand is selected from the group consisting of the sequences set forth in FIG. 2.
6. The nucleic acid ligand of claim 4 wherein said ligand is selected from the group consisting of: motif I (SEQ ID NOS.:4-10), motif II (SEQ ID NOS.:11-15), and motif III (SEQ ID NOS.:16-17), as shown in FIG. 3, wherein X is a non-conserved nucleotide position, X' is a base-pairing complement to X at that position in a helix, R is a purine and Y is a pyrimidine.
This is a continuation of application Ser. No. 07/953,694 filed on Sep. 29, 1992 which is a continuation-in-part of Ser. No. 07/714,131 filed Jun. 10, 1991, now U.S. Pat. No. 5,475,096, issued Dec. 12, 1995, entitled Nucleic Acid Ligands and U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned.
Described herein are methods for identifying and preparing ligands to a HIV-1 protein critical in the reproduction of the HIV-1 virus. Also included are the specific ligands identified pursuant to such methods. Specifically, nucleic acid ligands are described to the HIV-1 tat protein. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands for EXponential enrichment. Also included within the scope of this invention are modified nucleic acid ligands and mimetic ligands that are informed by the nucleic acid ligands identified herein.
Most proteins or small molecules are not known to specifically bind to nucleic acids. The known protein exceptions are those regulatory proteins such as repressors, polymerases, activators and the like which function in a living cell to bring about the transfer of genetic information encoded in the nucleic acids into cellular structures and the replication of the genetic material. Furthermore, small molecules such as GTP bind to some intron RNAs.
Living matter has evolved to limit the function of nucleic acids to a largely informational role. The Central Dogma, as postulated by Crick, both originally and in expanded form, proposes that nucleic acids (either RNA or DNA) can serve as templates for the synthesis of other nucleic acids through replicative processes that "read" the information in a template nucleic acid and thus yield complementary nucleic acids. All of the experimental paradigms for genetics and gene expression depend on these properties of nucleic acids: in essence, double-stranded nucleic acids are informationally redundant because of the chemical concept of base pairs and because replicative processes are able to use that base pairing in a relatively error-free manner.
The individual components of proteins, the twenty natural amino acids, possess sufficient chemical differences and activities to provide an enormous breadth of activities for both binding and catalysis. Nucleic acids, however, are thought to have narrower chemical possibilities than proteins, but to have an informational role that allows genetic information to be passed from virus to virus, cell to cell, and organism to organism. In this context nucleic acid components, the nucleotides, must possess only pairs of surfaces that allow informational redundancy within a Watson-Crick base pair. Nucleic acid components need not possess chemical differences and activities sufficient for either a wide range of binding or catalysis.
However, some nucleic acids found in nature do participate in binding to certain target molecules and even a few instances of catalysis have been reported. The range of activities of this kind is narrow compared to proteins and more specifically antibodies. For example, where nucleic acids are known to bind to some protein targets with high affinity and specificity, the binding depends on the exact sequences of nucleotides that comprise the DNA or RNA ligand. Thus, short double-stranded DNA sequences are known to bind to target proteins that repress or activate transcription in both prokaryotes and eukaryotes. Other short double-stranded DNA sequences are known to bind to restriction endonucleases, protein targets that can be selected with high affinity and specificity. Other short DNA sequences serve as centromeres and telomeres on chromosomes, presumably by creating ligands for the binding of specific proteins that participate in chromosome mechanics. Thus, double-stranded DNA has a well-known capacity to bind within the nooks and crannies of target proteins whose functions are directed to DNA binding. Single-stranded DNA can also bind to some proteins with high affinity and specificity, although the number of examples is rather smaller. From the known examples of double-stranded DNA binding proteins, it has become possible to describe some of the binding interactions as involving various protein motifs projecting amino acid side chains into the major groove of B form double-stranded DNA, providing the sequence inspection that allows specificity.
Double-stranded RNA occasionally serves as a ligand for certain proteins, for example, the endonuclease RNase III from E. coli. There are more known instances of target proteins that bind to single-stranded RNA ligands, although in these cases the single-stranded RNA often forms a complex three-dimensional shape that includes local regions of intramolecular double-strandedness. The amino-acyl tRNA synthetases bind tightly to tRNA molecules with high specificity. A short region within the genomes of RNA viruses binds tightly and with high specificity to the viral coat proteins. A short sequence of RNA binds to the bacteriophage T4-encoded DNA polymerase, again with high affinity and specificity. Thus, it is possible to find RNA and DNA ligands, either double- or single-stranded, serving as binding partners for specific protein targets. Most known DNA binding proteins bind specifically to double-stranded DNA, while most RNA binding proteins recognize single-stranded RNA. This statistical bias in the literature no doubt reflects the present biosphere's statistical predisposition to use DNA as a double-stranded genome and RNA as a single-stranded entity in the roles RNA plays beyond serving as a genome. Chemically there is no strong reason to dismiss single-stranded DNA as a fully able partner for specific protein interactions.
RNA and DNA have also been found to bind to smaller target molecules. Double-stranded DNA binds to various antibiotics, such as actinomycin D. A specific single-stranded RNA binds to the antibiotic thiostreptone; specific RNA sequences and structures probably bind to certain other antibiotics, especially those whose functions is to inactivate ribosomes in a target organism. A family of evolutionary related RNAs binds with specificity and decent affinity to nucleotides and nucleosides (Bass, B. and Cech, T. (1984) Nature 308:820-826) as well as to one of the twenty amino acids (Yarus, M. (1988) Science 240:1751-1758). Catalytic RNAs are now known as well, although these molecules perform over a narrow range of chemical possibilities, which are thus far related largely to phosphodiester transfer reactions and hydrolysis of nucleic acids.
Despite these known instances, the great majority of proteins and other cellular components are thought not to bind to nucleic acids under physiological conditions and such binding as may be observed is non-specific. Either the capacity of nucleic acids to bind other compounds is limited to the relatively few instances enumerated supra, or the chemical repertoire of the nucleic acids for specific binding is avoided (selected against) in the structures that occur naturally. The present invention is premised on the inventors' fundamental insight that nucleic acids as chemical compounds can form a virtually limitless array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and catalytic functions than those displayed in biological systems.
The chemical interactions have been explored in cases of certain known instances of protein-nucleic acid binding. For example, the size and sequence of the RNA site of bacteriophage R17 coat protein binding has been identified by Uhlenbeck and coworkers. The minimal natural RNA binding site (21 bases long) for the R17 coat protein was determined by subjecting variable-sized labeled fragments of the mRNA to nitrocellulose filter binding assays in which protein-RNA fragment complexes remain bound to the filter (Carey et al. (1983) Biochemistry 22:2601). A number of sequence variants of the minimal R17 coat protein binding site were created in vitro in order to determine the contributions of individual nucleic acids to protein binding (Uhlenbeck et al. (1983) J. Biomol. Structure Dynamics 1:539 and Romaniuk et al. (1987) Biochemistry 26:1563). It was found that the maintenance of the hairpin loop structure of the binding site was essential for protein binding but, in addition, that nucleotide substitutions at most of the single-stranded residues in the binding site, including a bulged nucleotide in the hairpin stem, significantly affected binding. In similar studies, the binding of bacteriophage Qβ coat protein to its translational operator was examined (Witherell and Uhlenbeck (1989) Biochemistry 28:71). The Qβ coat protein RNA binding site was found to be similar to that of R17 in size, and in predicted secondary structure, in that it comprised about 20 bases with an 8 base pair hairpin structure which included a bulged nucleotide and a 3 base loop. In contrast to the R17 coat protein binding site, only one of the single-stranded residues of the loop is essential for binding and the presence of the bulged nucleotide is not required. The protein-RNA binding interactions involved in translational regulation display significant specificity.
Nucleic acids are known to form secondary and tertiary structures in solution. The double-stranded forms of DNA include the so-called B double-helical form, Z-DNA and superhelical twists (Rich, A. et al. (1984) Ann. Rev. Biochem. 53:791-846). Single-stranded RNA forms localized regions of secondary structure such as hairpin loops and pseudoknot structures (Schimmel, P. (1989) Cell 58:9-12). However, little is known concerning the effects of unpaired loop nucleotides on stability of loop structure, kinetics of formation and denaturation, thermodynamics, and almost nothing is known of tertiary structures and three dimensional shape, nor of the kinetics and thermodynamics of tertiary folding in nucleic acids (Tuerk, C. et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364-1368).
A type of in vitro evolution was reported in replication of the RNA bacteriophage Qβ. Mills, D. R. et al. (1967) Proc. Natl. Acad. Sci USA 58:217-224; Levinsohn, R. and Spiegleman, S. (1968) Proc. Natl. Acad. Sci. USA 60:866-872; Levinsohn, R. and Spiegleman S. (1969) Proc. Natl. Acad. Sci. USA 63:805-811; Saffhill, R. et al. (1970) J. Mol. Biol. 51:531-539; Kacian, D. L. et al. (1972) Proc. Natl. Acad. Sci. USA 69:3038-3042; Mills, D. R. et al. (1973) Science 180:916-927. The phage RNA serves as a poly-cistronic messenger RNA directing translation of phage-specific proteins and also as a template for its own replication catalyzed by Qβ RNA replicase. This RNA replicase was shown to be highly specific for its own RNA templates. During the course of cycles of replication in vitro small variant RNAs were isolated which were also replicated by Qβ replicase. Minor alterations in the conditions under which cycles of replication were performed were found to result in the accumulation of different RNAs, presumably because their replication was favored under the altered conditions. In these experiments, the selected RNA had to be bound efficiently by the replicase to initiate replication and had to serve as a kinetically favored template during elongation of RNA. Kramer et al. (1974) J. Mol. Biol. 89:719 reported the isolation of a mutant RNA template of Qβ replicase, the replication of which was more resistant to inhibition by ethidium bromide than the natural template. It was suggested that this mutant was not present in the initial RNA population but was generated by sequential mutation during cycles of in vitro replication with Qβ replicase. The only source of variation during selection was the intrinsic error rate during elongation by Qβ replicase. In these studies what was termed "selection" occurred by preferential amplification of one or more of a limited number of spontaneous variants of an initially homogenous RNA sequence. There was no selection of a desired result, only that which was intrinsic to the mode of action of Qβ replicase.
Joyce and Robertson (Joyce (1989) in RNA: Catalysis, Splicing, Evolution, Belfort and Shub (eds.), Elsevier, Amsterdam pp. 83-87; and Robertson and Joyce (1990) Nature 344:467) reported a method for identifying RNAs which specifically cleave single-stranded DNA. The selection for catalytic activity was based on the ability of the ribozyme to catalyze the cleavage of a substrate ssRNA or DNA at a specific position and transfer the 3'-end of the substrate to the 3'-end of the ribozyme. The product of the desired reaction was selected by using an oligodeoxynucleotide primer which could bind only to the completed product across the junction formed by the catalytic reaction and allowed selective reverse transcription of the ribozyme sequence. The selected catalytic sequences were amplified by attachment of the promoter of T7 RNA polymerase to the 3'-end of the cDNA, followed by transcription to RNA. The method was employed to identify from a small number of ribozyme variants the variant that was most reactive for cleavage of a selected substrate.
The prior art has not taught or suggested more than a limited range of chemical functions for nucleic acids in their interactions with other substances: as targets for proteins that had evolved to bind certain specific olignocleotide sequences; and more recently, as catalysts with a limited range of activities. Prior "selection" experiments have been limited to a narrow range of variants of a previously described function. Now, for the first time, it will be understood that the nucleic acids are capable of a vastly broad range of functions and the methodology for realizing that capability is disclosed herein.
U.S. patent application Ser. No. 07/536,428 filed Jun. 11, 1990, of Gold and Tuerk, entitled Systematic Evolution of Ligands by Exponential Enrichment, and U.S. Pat. No. 5,475,096 issued Dec. 12, 1995 of Gold and Tuerk, entitled Nucleic Acid Ligands (See also WO 91/19813) describe a fundamentally novel method for making a nucleic acid ligand for any desired target. Each of these applications, collectively referred to herein as the SELEX Patent Applications, is specifically incorporated herein by reference.
The method of the SELEX Patent Applications is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether large or small in size.
The method involves selection from a mixture of candidates and step-wise iterations of structural improvement, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the method, termed SELEX herein, includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target pairs, amplifying the nucleic acids dissociated from the nucleic acid-target pairs to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired.
While not bound by a theory of preparation, SELEX is based on the inventors' insight that within a nucleic acid mixture containing a large number of possible sequences and structures there is a wide range of binding affinities for a given target. A nucleic acid mixture comprising, for example a 20 nucleotide randomized segment can have 420 candidate possibilities. Those which have the higher affinity constants for the target are most likely to bind. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands.
Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method may be used to sample as many as about 1018 different nucleic acid species. The nucleic acids of the test mixture preferably include a randomized sequence portion as well as conserved sequences necessary for efficient amplification. Nucleic acid sequence variants can be produced in a number of ways including synthesis of randomized nucleic acid sequences and size selection from randomly cleaved cellular nucleic acids. The variable sequence portion may contain fully or partially random sequence; it may also contain subportions of conserved sequence incorporated with randomized sequence. Sequence variation in test nucleic acids can be introduced or increased by mutagenesis before or during the selection/amplification iterations.
In one embodiment of the method of the SELEX Patent Applications, the selection process is so efficient at isolating those nucleic acid ligands that bind most strongly to the selected target, that only one cycle of selection and amplification is required. Such an efficient selection may occur, for example, in a chromatographic-type process wherein the ability of nucleic acids to associate with targets bound on a column operates in such a manner that the column is sufficiently able to allow separation and isolation of the highest affinity nucleic acid ligands.
In many cases, it is not necessarily desirable to perform the iterative steps of SELEX until a single nucleic acid ligand is identified. The target-specific nucleic acid ligand solution may include a family of nucleic acid structures or motifs that have a number of conserved sequences and a number of sequences which can be substituted or added without significantly effecting the affinity of the nucleic acid ligands to the target. By terminating the SELEX process prior to completion, it is possible to determine the sequence of a number of members of the nucleic acid ligand solution family.
A variety of nucleic acid primary, secondary and tertiary structures are known to exist. The structures or motifs that have been shown most commonly to be involved in non-Watson-Crick type interactions are referred to as hairpin loops, symmetric and asymmetric bulges, psuedoknots and myriad combinations of the same. Almost all known cases of such motifs suggest that they can be formed in a nucleic acid sequence of no more than 30 nucleotides. For this reason, it is often preferred that SELEX procedures with contiguous randomized segments be initiated with nucleic acid sequences containing a randomized segment of between about 20-50 nucleotides.
The SELEX Patent Applications also describe methods for obtaining nucleic acid ligands that bind to more than one site on the target molecule, and to nucleic acid ligands that include non-nucleic acid species that bind to specific sites on the target. The SELEX method provides means for isolating and identifying nucleic acid ligands which bind to any envisonable target. However, in preferred embodiments the SELEX method is applied to situations where the target is a protein, including both nucleic acid-binding proteins and proteins not known to bind nucleic acids as part of their biological function.
The HIV-1 tat protein activates transcription in the long terminal repeat (LTR) of the viral genome of HIV-1. See, Cullen et al. (1989) Cell 58:423-426. The mechanism of activation is unclear or at least controversial, but requires that the transcribed RNA contain a specific hairpin structure with a trinucleotide bulge (called TAR). The natural TAR RNA and the site of tat interaction is shown in FIG. 1. A small basic domain of the tat protein has been shown to interact directly with the TAR RNA sequence. See, Weeks et al. (1990) Science 249:1281-1285; Roy et al. (1990) Genes Dev. 4:1365-1373; Calnan et al. (1991a) Genes Dev. 5:201-210. Arginines within this basic domain are apparently crucial to the interaction. See, Calnan et al. (1991a) supra; Subramanian et al. (1991) EMBO 10:2311-2318; Calnan et al (1991b) Science 252:1167-1171. Arginine alone is specifically bound by the TAR RNA sequence and may compete for tat protein binding. See, Tao et al. (1992) Proc. Natl. Acad. Sci. USA 89:2723-2726; Puglisi et al. (1992) Science 257:76-80.
Tat--TAR interactions alone are insufficient to support transactivation; presumably a cellular factor --a 68 kD loop binding protein--is required for cooperative binding with the tat protein to TAR, and subsequent in vivo or in vitro transactivation. See, Marciniak et al. (1990a) Proc. Natl. Acad. Sci. USA 87:3624-3628; Marciniak et al. (1990b) Cell 63:791-802. Overexpression of the TAR sequence in retrovirally transformed cell lines renders them highly resistant to HIV-1 infections. See, Sullenger et al. (1990) Cell 63:601-608.
The present invention includes methods for identifying and producing nucleic acid ligands and the nucleic acid ligands so identified and produced.
Nucleic acid sequences are provided that are ligands to the HIV-1 tat protein. More specifically, RNA sequences have been identified that are capable of binding to the tat protein. Included within the invention are the nucleic acid ligand solutions shown in FIGS. 2 and 3.
Further included in this invention is a method of identifying nucleic acid ligands and ligand solutions to the HIV-1 tat protein comprising the steps of a) preparing a candidate mixture of nucleic acids; b) partitioning between members of said candidate mixture on the basis of affinity to the tat protein; and c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to the tat protein.
The present invention also includes modified nucleotide sequences based on the nucleic acid ligand solutions identified herein and mixtures of the same.
FIG. 1 depicts the natural RNA sequence (SEQ ID NO:20) (or TAR RNA) from HIV-1 with which the tat protein interacts. The boxed region of the sequence identifies those nucleotides that have been found to be important in the tat-TAR interaction.
FIG. 2 lists the sequences of ligands isolated by the present invention as nucleic acid ligands to the HIV-1 tat protein (SEQ ID NO:4-19). The sequences are grouped according to common secondary structures and primary sequence in three motifs (I, II, and III). Inverted repeat sequences that predict RNA helices are shown with arrows. The regions of primary sequence homology within each motif are outlined with dashed boxes. The boundaries of the sequence information essential for high affinity binding is indicated by a solid-lined box. Sequences 1 and 17 do not fit into any of the three identified motifs.
FIG. 3 depicts a schematic diagram of the consensus secondary structure and primary sequence of each of the ligand motifs given in FIG. 2 (SEQ ID NO:4-19). X indicates non-conserved nucleotide positions. X' indicates a base-pairing complement to X at that position in a helix, R indicates purine and Y pyrimidine. The dashed line in motif III indicates a variable number of nucleotides at that portion of the loop.
FIG. 4 is the tat protein concentration-dependent binding of selected ligand RNAs from FIG. 2, and the 40n RNA candidate mixture, to nitrocellulose filters.
This application is an extension and an application of the method for identifying nucleic acid ligands referred to as SELEX. The SELEX method is described in detail in U.S. Pat. No. 5,475,096 issued Dec. 12, 1995 entitled Nucleic Acid Ligan, continuation-in-part of application Ser. No. 07/536,428 filed Jun. 11, 1990 entitled Systematic Evolution of Ligands by EXponential Enrichments, now abandoned. The full text of these applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference.
In its most basic form, the SELEX process may be defined by the following series of steps:
1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: a) to assist in the amplification steps described below; b) to mimic a sequence known to bind to the target; or c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
2) The candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and the nucleic acids having the strongest affinity for the target.
3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.
4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
The SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for the preparation of the initial candidate mixture; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixtures. The SELEX Patent Applications also describe ligand solutions obtained to a number of target species, including both protein targets wherein the protein is and is not a nucleic acid binding protein.
SELEX delivers high affinity ligands of a target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to a specific target, the HIV-1 tat protein. In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligand solution to the HIV-1 tat protein are described. FIG. 2 lists the nucleic acids that were sequenced after 10 iterations of the SELEX process.
FIG. 1 shows the naturally occurring TAR sequence that has been found to be a natural ligand to the tat protein. The specific site of interaction between the tat protein and the TAR sequence has been determined, and is also identified in FIG. 1.
The sequences presented in FIG. 2 are grouped into three "motifs". Each of these motifs represents a nucleic acid ligand solution to the HIV-1 tat protein. Regions of primary sequence conservation within each motif are boxed with dashed lines. Motifs I and II contain a common structure that places conserved sequences (those sequences found in all or most all of the nucleic acid sequences that make up the given motif) in a bulge flanked by helical elements. The primary sequence conservation--which is mainly in the single stranded domains of each bulge--are also similar between motifs I and II. The third motif (III) is characterized by a large loop. The three motifs are depicted schematically in FIG. 3. There is no apparent similarity between the nucleic acid ligands identified herein and the TAR sequence given in FIG. 1.
A boundary analysis determination was performed on one of the ligand sequences in motif III. The boundaries of recognition are indicated by a solid-lined box in FIG. 2. The boundary determination was performed according to previously described techniques. See, Tuerk et al. (1990) J. Mol. Biol. 213:749-761; Tuerk et al. (1990) Science 249:505-510.
In FIG. 4, the binding affinities of sequences 7 (motif I), 24 (motif II), 29 (motif II), 31 (motif III) and the original candidate mixture are depicted. As can be seen, members from each of the nucleic acid ligand solution motifs have increased affinity to the tat protein relative to the candidate mixture of nucleic acids. Each of the ligands exhibits a significantly greater affinity to the tat protein relative to the TAR sequence.
In order to produce nucleic acids desirable for use as a pharmaceutical, it is preferred that the nucleic acid ligand 1) binds to the target in a manner capable of achieving the desired effect on the target; 2) be as small as possible to obtain the desired effect; 3) be as stable as possible; and 4) be a specific ligand to the chosen target. In most, if not all, situations it is preferred that the nucleic acid ligand have the highest possible affinity to the target.
In copending and commonly assigned U.S. Pat. application Ser. No. 07/964,624, methods are described for obtaining improved nucleic acid ligands after SELEX has been performed. This application, entitled Methods of Producing Nucleic Acid Ligands is specifically incorporated herein by reference. Included in this application are the following methods relating to: Assays of ligand effects on target molecules; Affinity assays of the ligands; Information boundaries determination; Quantitative and qualitative assessment of individual nucleotide contributions to affinity via secondary SELEX, nucleotide substitution, and chemical modification experiments; and structural determination. The present invention includes improvements to the nucleic acid ligand solution derived according to these procedures.
This invention includes the specific nucleic acid ligands shown in FIG. 2 and the nucleic acid ligand solutions as depicted schematically in FIG. 3. The scope of the ligands covered by this invention extends to all ligands to the tat protein identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are 1) substantially homologous to and that have substantially the same ability to bind the tat protein as the specific nucleic acid ligands shown in FIG. 2 or that are 2) substantially homologous and that have substantially the same ability to bind the tat protein as the nucleic acid ligand solutions shown in FIG. 3. By substantially homologous, it is meant, a degree of primary sequence homology in excess of 70% most preferably in excess of 80%. Substantially the same ability to bind the tat protein means that the affinity is within two orders of magnitude of the affinity of the substantially homologous sequence described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence--substantially homologous to those specifically described herein--has substantially the same ability to bind the tat protein.
A review of motifs I, II and III, and the binding curves shown in FIG. 4, show that sequences that have little or no primary sequence homology may still have substantially the same ability to bind the tat protein. If one assumes that each of these motifs of ligands binds the same binding site of the tat protein, it is clear that binding is controlled by the secondary or tertiary structure of the nucleic acid ligand. Certain primary structures--represented by motifs I, II and III herein--are apparently able to assume structures that appear very similar to the binding site of the tat protein. For these reasons, the present application also includes nucleic acid ligands that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind the tat protein as the nucleic acid ligands shown in FIG. 2 or FIG. 3. Wherein substantially the same structure includes all nucleic acid ligands having the common structural elements of motifs I, II and III that lead to the affinity to the tat protein.
This invention also includes the ligands as described above, wherein certain chemical modifications have been made in order to increase the in vivo stability of the ligand or to enhance or mediate the delivery of the ligand. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions of a given RNA sequence. See, e.g., Cook et al. PCT Application WO 9203568; U.S. Pat. No. 5,118,672 of Schinazi et al.; Hobbs et al. Biochem. 12:5138 (1973); Guschlbauer et al. Nucleic Acids. Res. 4:1933 (1977); Schibaharu et al. Nucl. Acids. Res. 15:4403 (1987); Pieken et al. Science 253:314 (1991).
The nucleic acid ligands and nucleic acid ligand solutions to the HIV-1 tat protein described herein are useful as pharmaceuticals and as part of gene therapy treatments. According to methods known to those skilled in the art, the nucleic acid ligands may be introduced intracellularly into cells infected with the HIV virus, where the nucleic acid ligand will compete with the TAR sequence for the tat protein. As such, transcription of HIV genes can be prevented.
tat protein was purchased from American Bio-Technologies, Inc. Templates for in vitro transcription were produced by PCR using the following oligonucleotides: ##STR1## SELEX rounds were conducted as described in Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA, in press, and in the SELEX Applications, under the following conditions: Binding reactions were done with 13 nanomolar tat protein and 1.3 micromolar RNA in a volume of 2 mls for rounds 1 and 2, and 6.5 nanomolar tat protein and 0.65 micromolar RNA in 4 mls for rounds 3-10.
RNA synthesis. In vitro transcription with oligonucleotide templates was conducted as described by Milligan et al. (1987) Nucl. Acid. Res. 15:8783-8798. All synthetic nucleic acids were made on the Applied Biosystems model 394-08 DNA/RNA synthesizer using standard protocols. Deoxyribonucleotide phosphoramidites and DNA synthesis solvents and reagents were purchased from Applied Biosystems.
Affinity assays with labeled RNA and HIV-1 tat protein. Model RNAs for refinement of the 5' and 3' boundaries and for determination of the effect of substitutions were labeled during transcription with T7 RNA polymerase as described in the SELEX Applications, except that α-32 P-ATP was used, in reactions of 0.5 mM C, G, and UTP with 0.05 mM ATP. All RNA-protein binding reactions were done in a "binding buffer" of 200 mM KOAc, 50 mM Tris-Hcl pH 7.7, 10 mM dithiothreitol. RNA and protein dilutions were mixed and stored on ice for 30 minutes then transferred to 37� C. for 5 minutes. In binding assays the reaction volume was 60 ul of which 50 ul was assayed. Each reaction was suctioned through a pre-wet (with binding buffer) nitrocellulose filter and rinsed with 3 mls of binding buffer after which it was dried and counted for assays or subjected to elution and assayed for chemical modification. In comparisons of binding affinity, results were plotted and the protein concentration at which half-maximal binding occurred (the approximate Kd in conditions of protein excess) was determined graphically. The results of the binding assays are given in FIG. 4.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 20(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 48 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCGAAGCTTAA TACGACTCACTATAGGGAGCTCAGAATAAACGCTCAA48(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 87 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GCCGGATCCGGGCCTCAT GTCGAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN60NNNNTTGAGCGTTTATTCTGAGCTCCC87(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GCCGGATCCGGGCCTCATGTCGAA24(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 51 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:AAGCCUCAGUAAGGCAACGGAAUCCGCAAGAGGAUGGACCACUUCGACAUG51(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 55 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GCUCAACACGAAGGAAACGGAGGGAAUCUUGAAGAACCCGGACCACUUCGACAUG55(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:CUCAAGCGGAAUGGAAACGGAGCCAUCAACAAGCUGGCGGACCACAUUCGACAU54(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 53 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:UCAACAAACGAAGGAAACGGAUGACCCAAGCAGGUCAGGACCACUUCGACAUG53(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:CGCUCAAACGAAGGAAACGGAUGCGGACAUAUGUGCCGCAGGACCACUUCGACAUG56(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:AAACACAUCGAAGGUAACGGAGCGAAAAGAACGCGGACCACUUCGACAUG50(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:A ACGCUCAAGCGGGCAACGGAGUCCUGAACGGACGGACCACCGCAAGAAU50(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:CUCAAGCG GACAGUAAACGACCCACGAUUGCGAUGGGGACACAAGUUCGACAUG54(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CUCAAAAGAAGGGU CAACGACCAACUCCUACAGUUGGGACACAACUUCGACAUG54(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 49 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CAAUGAAUUGGAGUAAACGA CUCACCAAUGAGGACACAACCAAUUCGAC49(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 56 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GCUCAAGGCGAUGGAAACGACGGAAAA GAGAAAAGUACGGACACAAUUCGACAUGA56(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 55 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:CUCAAUCGAACGGUUAACGACCUCGAACACAAG GGGGGCACAAAAUUCGACAUGA55(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 55 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:AAGAGAAUCUAACGAGACGUAAGCCGUCGGAGAAUGAGAC GAUUCGACAUGAGGC55(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 53 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:AGUGAGGACUAAUGAGGCGUACGACCGGAGAUUGAGACGUCUUCGA CAUGAGG53(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:CGCUCAAGGCGACGGGACUGCAAGCAUGGAGCUAACGAGAAAAUUGCUUCGA CA54(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 54 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:CGCUCAACCUGGAGAGGAUCGCUGGCGGGCUUGAUCCCCAGAUCAAAUUCGACA 54(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CCAGAUCUGAGCCUGGGAGCUCUCUGG27__________________________________________________________________________
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